The present invention is in the field of pharmacogenomics, and is primarily directed to biallelic markers that are located in or in the vicinity of genes, which have an impact on the metabolism of xenobiotics such as drugs and the uses of these markers. The present invention encompasses methods of establishing associations between these markers and a phenotype such as drug response, toxicity and susceptibility to disease. The present invention also provides means to determine the genetic predisposition of individuals to such drug responses, toxicity and diseases.
To assess the origins of individual variations in drug response, pharmacogenomics uses the genomics technologies to identify polymorphisms within genes associated with drug response. In this respect, there are three main categories of genes that may theoretically be expected to be associated with drug response, namely genes linked with the targeted disease, genes related to the drug""s mode of action and genes involved in the drug""s metabolism. Among these genes of pharmacogenomic importance, genes coding for drug-metabolizing enzymes have a central role.
Drug Metabolism
Drug-metabolizing enzymes are important determinants of drug disposition, safety and efficacy. The enzyme systems involved in the metabolism and the subsequent elimination from the body of environmental chemicals, food toxins and drugs are mainly localized in the liver, although every tissue examined has some metabolic activity.
In order to produce its characteristic effects, a given drug must be present in appropriate concentrations at its sites of action. The absorption, distribution, biotransformation and excretion of a drug all involve its passage across cell membranes. The lipophilic characteristics of drugs that promote their passage through biological membranes and subsequent access to their site of action reduce their elimination from the body. Renal excretion of unchanged drug plays only a modest role in the overall elimination of most therapeutic agents, since lipophilic compounds filtered through the glomerulus are largely reabsorbed through the tubular membranes. Biotransformation of drugs into more hydrophilic metabolites plays a major role in the termination of their biological activity and their elimination from the body. In general, biotransformation reactions generate more polar, inactive metabolites that are readily excreted from the body. However in some cases, metabolites with potent biological activity or toxic properties are generated and may result in adverse side effects. Metabolic biotransformation of drugs can be classified as either Phase I fumctionalization reactions or Phase II biosynthetic reactions. Phase I reactions introduce or expose a functional group on the parent compound, and generally result in the loss of pharmacological activity although there are some examples of retention or enhancement of activity. Phase II conjugation reactions lead to the formation of a covalent linkage between a functional group on the parent compound with glucuronic acid, sulfate, glutathione, amino acids or acetate. These highly polar conjugates are generally inactive and are excreted rapidly in the urine and feces. Within a given cell, most drug metabolizing Phase I enzymes are located primarily in the endoplasmic reticulum, while the Phase II conjugation enzyme systems are mainly cytosolic. In some cases, drugs biotransformed through a Phase I reaction in the endoplasmic reticulum are further metabolized by conjugation in the cytosolic fraction of the same cell (Hardman J. G., Goodman, Gilman A., Limbird L. E.; Goodman and Gilman""s The Pharmacological Basis of Therapeutics, 9th edition, McGraw-Hill, N.Y., 1996).
Enzymes Involved in the Biotransformation of Xenobiotics
Besides being involved in the biotransformation of drugs, drug-metabolizing enzymes are also involved in the metabolism of xenobiotics (foreign compounds) as well as in the metabolism of endogenous compounds including steroids, vitamins and fatty acids. Foreign compounds include therapeutic agents, carcinogens, plant metabolites, environmental pollutants, foodstuffs and other dietary components as well as industrial chemicals. The biotransformation of foreign compounds (xenobiotics) is often regarded as detoxification because it usually converts compounds into more water-soluble, readily excreted substances. This tends to decrease the exposure of the organism to the compound and therefore tends to decrease toxicity. However, in some cases the reverse occurs and a metabolite is produced which is more toxic than the parent compound. For example, drug-metabolizing enzymes may activate some carcinogens, and interindividual differences in cancer susceptibilities, have been linked to polymorphisms in drug-metabolizing enzymes. There are many factors, which affect biotransformation and toxicity, such as the dose, availability of cofactors and the relative activity of the various drug-metabolizing enzymes. There may also be several competing pathways of metabolismxe2x80x94some leading to detoxification others to toxicity. Factors, such as genetic factors or environmental factors, which influence the balance between these competing pathways, will also determine the eventual toxicity.
As mentioned above, the metabolic conversion of drugs and other xenobiotics is enzymatic in nature. The enzyme systems involved in the biotransformation of drugs are localized in the liver, although every tissue examined has some metabolic activity. Other organs with significant metabolic capacity include the kidneys, gastrointestinal tract, skin and lungs. Following non-parenteral administration of a drug, a significant portion of the dose may be metabolically inactivated in either the liver or intestines before it reaches the systemic circulation. This first-pass metabolism significantly limits the oral availability of highly metabolized drugs.
Cytochrome P450
The cytocbrome P450 enzyme family is the major catalyst of biotransformation reactions. Since its origin, the cytochrome P450 gene family has diversified to accommodate the metabolism of a growing number of environmental chemicals, food toxins and drugs. The resulting superfamily of enzymes catalyzes a wide variety of oxidative and reductive reactions and has activity towards a chemically diverse group of substrates. Cytochrome P450 enzymes are heme-containing membrane proteins localized in the smooth endoplasmic reticulum of numerous tissues. Oxidative reactions catalyzed by the microsomal monooxygenase system require the cytochrome P450 hemoprotein, NADPH-cytochrome P450 reductase, NADPH, and molecular oxygen. Oxidative biotransformations catalyzed by cytochrome P450 monooxygenases include aromatic and side chain hydroxylation, N-, O-, S- dealkylation, N-oxidation, sulfoxidation, N-hydroxylation, deamination, dehalogenation, and desulfuration. Cytochrome P450 enzymes also catalyze a number of reductive reactions, generally under conditions of low oxygen tension. The only common structural feature of the diverse group of xenobiotics oxidized by cytochrome P450 enzymes is their high lipid solubility.
Twelve cytochrome P450 gene families have been identified in human beings, and a number of distinct cytochrome P450 enzymes often exist within a single cell. The cytochrome P450 1, 2 and 3 families (CYP1, CYP2, CYP3) encode the enzymes involved in the majority of all drug biotransformations, while the gene products of the remaining cytochrome P450 families are important in the metabolism of endogenous compounds such as steroids and fatty acids. CYP1A2 gene expression may play an important role in individual risk of environmental toxicity or cancer. CYP1A2 substrates include clinically important drugs such as imipramine, propranolol, paracetamol, clozapine, theophyline, caffeine and acetaminophen. CYP1A2 is also involved in the conversion of heterocyclic amines and arylamines to their proximal carcinogenic and mutagenic forms, as well as in the metabolism of endogenous substances including estradiol and uroporphyrinogen III. Interindividual differences in susceptibility to arylamine- and heterocyclicamine-induced cancers have been linked to CYP1A2 polymorphism. CYP2C8 appears to be responsible for retinol and retinoic acid metabolism and actively catalyzes benzphetamine N-demethylation. CYP2C9 catalyzes the hydroxylation of tolbutamide, a hypoglycemic agent used in the treatment of type II diabetes mellitus, and one allelic variant of CYP2C9 accounts for the occurrence of poor metabolizers of tolbutamide. CYP2C9 may also have an important role in terminating the anti-coagulant activity of warfarin. Widespread interindividual differences in the response to warfarin have been recognized. Such variability is particularly important for drugs such as warfarin which have narrow therapeutic indices (Steward D. J. et al., Pharmacogenetics, 7:361-367, 1997). CYP2C9 is further involved in the oxidation of tielinic acid and several non-steroidal anti-inflammatory agents. The oxidative metabolism through CYP2C9 of tilenic acid can result in the emergence of a drug induced autoimmune hepatitis. CYP3A4 is involved in the biotransformation of a majority of drugs and is expressed at significant levels extrahepatically. It is now recognized that extensive metabolism by CYP3A4 in the gastrointestinal tract is a significant factor contributing to the poor oral availability of many drugs (first-pass metabolism). Barbiturates, certain steroids and macrolide antibiotics can induce this enzyme. It appears to play a central role in the metabolism of the immunosuppressive cyclic peptide cyclosporin A as well as macrolide antibiotics, such as erythromycin.
Flavin-Containing Monooxygenases (FMOs)
The mammalian flavin-containing monooxygenases (FMOs) are microsomal enzymes that catalyze the NADPH-dependent oxygenation of a wide variety of drugs and other xenobiotics that possess a soft nucleophilic heteroatom, typically a nitrogen, sulfur, phosphorus or selenium atom. Of special clinical interest is the oxidation of trimethylamine in the liver by the FMO, because its deficiency causes the xe2x80x9cFish Odor Syndrome.xe2x80x9d Drugs oxidized by FMOs include, among others, antidepressant, antipsychotic-neuroleptic, antihypertensive drugs. FMOs have been implicated in the detoxification but also in the metabolic activation of several different environmental toxins and carcinogens.
Unlike all other known oxidases and monooxygenases, among which the well-studied cytocbrome P450 monooxygenases, FMOs have the unique property of forming a stable enzyme intermediate in the absence of an oxygenatable substrate. Because the energy for catalysis is already present in the FMO enzyme before contact with the potential substrate, the fit of the substrate does not need to be as stringent as with the other enzymes. This feature, unique to FMOs among monooxygenases, is responsible for the wide range of substrates accepted by FMOs (including tertiary and secondary alkyl- and arylamines, many hydrazines, thiocarbamides, thioamides, sulfides, disulfides, thiols, among others), and determines that any soft nucleophilic xenobiotic accessible to the active enzyme will probably be oxidized by FMO in vivo. Although some FMO substrates are oxidized to less active derivatives, several soft nucleophiles are metabolized to highly reactive and potentially toxic intermediates.
The FMOs represent a multigene family. Five distinct mammalian FMO isoenzymes have been identified and cloned from various animal and human tissues: FMO1, FMO2, FMO3, FMO4 and FMO5. Human FMO2 and human FMOX were cloned and sequenced by the inventors as described in PCT Publication WO 9824914. FMOX represents a new member of the FMO gene family not previously identified in mammals. Tissue specificity and activities of the different FMOs have been thoroughly characterized. FMO1 is known to be expressed in the human kidney but is absent from the liver. In man the enzyme is subject to developmental regulation. FMO2 is predominantly expressed in lung of all mammalian species tested. FMO3 was isolated from human liver, and accounts for the majority of FMO expressed in adult human liver.
Many of the FMO substrates may also be oxidized by the cytochrome P450 monooxygenases. However, the final oxidation products are usually different, and the nitrogen of a specific compound is rarely N-oxygenated by both types of monooxygenases. Today, a large number of drugs in human clinical trials contain a nitrogen, sulfur, phosphorous or some other nucleophilic functionality. Of the two major monooxygenase systems considered to be responsible for heteroatom-containing chemical and drug oxidative metabolism (CYP 450 and FMO), relatively little is known concerning the role of the FMO in human drug metabolism. Yet, given the wide range of substrates potentially oxidized by FMOs, this class of monooxygenases seems to represent a major determinant of drug safety and efficacy.
Uridine Diphosphate Glucoronosyl Transferase (UGTs)
Glucoronidation is a major detoxification pathway of Phase II metabolism that is catalyzed by the UDP-glucuronosyl transferase family of enzymes. Glucuronidation is quantitatively the most important conjugation reaction. Members of this enzyme family catalyze the conjugation of numerous endogenous substances of widely differing structures such as bilirubin, steroid hormones and fat-soluble vitamins. In general, xenobiotics become substrates for glucoronidation by first passing through Phase I metabolism, but many compounds do not require this step because they already possess reactive functionalities (e.g. hydroxyl, carboxyl, amino, sulfhydryl etc.) that are direct targets for glucuronosyl transferase. The human UGT genes appear to have evolved by a series of gene-duplication and gene-conversion events resulting in the emergence of a diversity of isoforms. They are divided into two families, UGT1 which is known to have bilirubin and phenol as substrates, and UGT2 which is known to have steroid, bile, and odorant as substrates, with these two families located on different chromosomes. The UGT2 family is divided into subfamilies UGT2A and UGT2B. The UGTs have different but sometimes overlapping substrate specificities. They catalyze the transfer of an activated glucuronic acid molecule to aromatic and aliphatic alcohols, carboxylic acids, amines and free sulfhydryl groups of both exogenous and endogenous compounds, to form O- N- and S-glucuronide conjugates. The increased water solubility of the glucuronide conjugates promotes their elimination in the urine or bile. In addition to high levels of expression in the liver, UGTs are also found in the kidney, intestine, brain and skin. Glucoronidation constitutes, from a general point of view, a reaction of detoxification and elimination. It generally leads to the formation of inactive metabolites and therefore, glucoronidation can dramatically modify the pharmacological activity of a drug. Moreover, UGTs play a major role in the elimination of nucleophilic metabolites of carcinogens, such as phenols and quinols of polycyclic aromatic hydrocarbons. In this way they prevent their further oxidation to electrophiles, which may react with DNA, RNA or protein. On the other hand, glucoronidation of certain compounds facilitates metabolic activation. Aromatic amines are some of the most studied examples of the role glucoronidation plays in metabolic activation of carcinogens. Glucoronidation has also been implicated in adverse drug reactions of certain carboxylic drugs, which resulted in a toxic immunological response. Glucoronidation although generally a detoxification reaction, may occasionally be involved in increasing toxicity.
Glutathione Conjugation and Further Metabolism
Glutathione is a tripepetide (xcex3-glutamylcysteinylglycine, GSH) found in high concentrations in most mammalian tissues, but especially in the liver. Glutathione has several functions including roles in metabolism, transport and catalysis. Glutathione is also important for the maintenance of the thiol moieties of proteins and for the maintenance of the reduced form of other molecules such as cysteine, coenzyme A, and antioxidants such as ascorbic acid; it is also used in the formation of deoxyribonucleic acids (Anderson M. E., Advances in Pharmacology, 38: 65-74, 1997). Glutathione has a major protective role in the body, as it is the major cellular antioxidant. GSH can react non-enzymatically with reactive oxygen species (ROS) and thereby protect the cell from oxidative damage. ROS have been widely implicated in the pathology of numerous diseases such as arteriosclerosis, rheumatoid arthritis, cancer, AIDS, adult respiratory distress syndrome and Parkinson""s disease.
Moreover, conjugation with the tripeptide glutathione represents a major detoxification pathway for xenobiotics including drugs and carcinogens. Glutathione may react either chemically or in enzyme catalyzed reactions with a variety of compounds, which are reactive electrophilic metabolites produced in Phase I reactions. The glutathione S-transferase enzymes (GSTs) that catalyze these reactions are members of a multigene family and are expressed in virtually all tissues. Glutathione conjugates are cleaved to cysteine derivatives and subsequently are acetylated by a series of enzymes located primarily in the kidney to give N-acetylcysteine conjugates collectively referred to as mercapturic acids. Mercapturic acid derivatives are the ultimate metabolites excreted in the urine. This is a particularly important route of Phase II metabolism from the toxicological point of view, as it is often involved in the removal of reactive intermediates. Xenobiotics that act as substrates for the glutathione S-transferases (GSTs) fall into four broad categories: electropilic carbon, nitrogen, sulfur and oxygen. Examples of substrates for glutathione S-transferases include aromatic, heterocyclic, alicyclic and aliphatic epoxides; aromatic halogen and nitro compounds; alkyl halides; and unsaturated aliphatic compounds (Ballantyne, B., Marrs T. and Turner P., General and Applied Toxicology, Stockton Press, New York, 1993). The GSTs are also involved in the metabolism of endogenous molecules such as the leukotrienes. As mentioned above, many of the enzymes involved in xenobiotic metabolism are also involved in specific aspects of the metabolism of normal cellular biochemical constituents. Leukotrienes are important mediators and modulators of the inflammatory reaction and contribute to a number of physiological and pathological processes. Moreover, the GSTs are also capable of directly binding hydrophobic compounds such as heme, bilirubin, and steroids, which may enable them to serve as intracellular storage and transport proteins for biological substances with limited water solubility. By their catalytic activity and their capacity for binding, the GSTs provide the cell with mechanism to protect itself from the noxious effects of various xenobiotics and endogenous substances. Further, GSTs may undergo amplification in tumors and may thereby be implicated in drug resistance in cancer chemotherapy. GSTs are mostly cytosolic although, more recently, microsomal GSTs have been identified. Human microsomal GST II (MGST II) is a member of the microsomal glutathione S-transferase family. This enzyme catalyzes the production of LTC4 (leukotriene C4) from LTA4 (leukotriene A4) and reduced glutathione. Leukotrienes are derived from arachidonic acid and related fatty acids. Metabolites of arachidonic acid have been collectively termed eicosanoids, the principal eicosanoids are prostaglandins, thromboxanes and leukotrienes (LT). Eicosanoids are among the most important chemical mediators and modulators of the inflammatory reaction and contribute to a number of physiological and pathological processes (see Hardman J. G., Goodman, Gilman A., Limbird L. E.; Goodman and Gilman""s The Pharmacological Basis of Therapeutics, 9th edition, McGraw-Hill, N.Y., 1996). The ability to mount an inflammatory response is essential for survival in the face of environmental pathogens and injury, although in some situations and diseases the inflammatory response may be exaggerated and sustained for no apparent beneficial reason. This is the case in numerous chronic inflammatory diseases and allergic inflammation. Acute allergic inflammation is characterized by increased blood flow, extravasation of plasma and recruitment of leukocytes. These events are triggered by locally released inflammatory mediators including eicosanoids and more particularly leukotrienes. The participation of arachidonic acid metabolism in inflammatory diseases such as rheumatoid arthritis, asthma and acute allergy is well established. Pathological actions of leukotrienes are best understood in terms of their roles in immediate hypersensitivity and asthma. LTC4 and LTD4 are potent bronchoconstrictors, they act principally on smooth muscle in peripheral airways and are a 1000 times more potent than histamine both in vitro and in vivo. They also stimulate bronchial mucus secretion and cause mucosal edema. A complex mixture of chemical messengers is released when sensitized lung tissue is challenged by the appropriate antigen. Various prostaglandins and leukotrienes are prominent components of this mixture. Response to the leukotrienes probably dominates during allergic constriction of the airway. A particularly important role for the cysteinyl-leukotrienes (LTC4, LTD4, and LTE4) has been suggested in pathogenesis of asthma, which is now recognized as a chronic inflammatory condition. They are potent spasmogens causing a contraction of bronchiolar muscle and an increase in mucus secretion. An increased LTC4 formation has also been reported in leukocytes from patients with chronic myelogenous leukemia (Stenke et al., Acta Oncologica, 27:803-805, 1987) and in experimental glomerulonephritis (Petric et al., Biochim. Biophys. Acta, 1254:207-215, 1995).
Moreover, MGST II has the capacity to conjugate other compounds such as 1-chloro-2,4 dinitrobenzene with glutathione and may be involved in a general metabolic system for detoxifying fatty acid epoxides (Jakobsson et al., Journal of Biological Chemistry, 271:22203-22210, 1996).
The resulting glutathione conjugate usually undergoes further metabolism, which involves first a removal of the glutamyl residue, catalyzed by xcex3-glutamyltransferase (GGT). In addition to catalyzing the initial step in the conversion of glutathione-conjugated compounds to mercapturic acids, GGT also converts LTC4 to LTD4. Interestingly, expression of GGT is often increased in cancerous tissues.
Renal dipeptidase is also implicated in the renal metabolism of glutathione and its conjugates including conjugated xenobiotics and endogenous molecules such as Leukotriene D4. Pharmacologically it is an important enzyme, for it is responsible for hydrolysis of some xcex2-lactam antibiotics such as penem and carbapenem.
The effectiveness of the GSTs and therefore of detoxification by glutathione conjugation in general as well as the ability of the cell to resist to oxidative stress, are strongly influenced by the availability of reduced glutathione. Reduction of oxidized glutathione and de novo synthesis of glutathione, are both completely dependent on NADPH. Glutathione reductase (GSHR) maintains high levels of reduced glutathione in the cytosol in an NADPH dependent reaction. Reduced glutathione is synthesized de novo in the cytosol of most cells via the xcex3-glutamyl cycle; a series of tightly controlled, enzyme catalyzed reactions. The first and second step in the de novo glutathione biosynthesis are catalyzed by xcex3-glutamylcysteine synthetase (GLCL) and glutathione synthase (GSHS) respectively. Deficiencies in xcex3-glutamylcysteine synthetase and GSH synthetase are associated with hemolytic anemia and impaired central nervous system function. Glucose 6-phosphate Dehydrogenase (G6PDH), Phosphogluconate Dehydrogenase (PGDH) and Malate Dehydrogenase: Generation of NADPH
NADPH (nicotinamide adenine dinucleotide phosphate) serves as an electron donor in reductive biosyntheses. In the pentose phosphate pathway, NADPH is generated when glucose 6-phosphate is oxidized to ribose 5-phosphate. G6PDH and PGDH are key enzymes of the pentose phosphate pathway and directly lead to the generation of NADPH. Another major source of NADPH is the oxidative decarboxylation of malate by malic enzyme.
NADPH may be used in anabolic processes such as fatty acid biosynthesis. One of the major functions of malic enzyme may be supplying NADPH to the cytosol for the synthesis of fatty acids from acetyl CoA (coenzyme A). Further, the cytochrome P450 system is dependent on NADPH. As mentioned above, availability of NADPH is also critical for the reduction of glutathione. The connection between generation of NADPH and reduction of glutathione is clearer in tissues that have limited glycolytic metabolism, e.g. the lens and the erythrocyte. Thus the viability of the erythrocyte depends on glutathione, kept reduced by this pathway. Moreover, factors that influence the availability of reduced glutathione drastically alter the effectiveness of glutathione S-transferases therefore also affecting drug metabolism. Under most conditions saturating levels of NADPH are provided to the cell. However, certain conditions can stress the ability of the cell to provide NADPH and it may become rate limiting.
G6PDH and PGDH are present in most cells and tissues. They serve as the key enzymes of the pentose phosphate pathway that control the flow of carbon through the pathway and produce reducing equivalents as NADPH to meet cellular needs for reductive biosynthesis and to maintain the redox state of the cell at physiological levels. Deficiency of G6PDH and PGDH leads to decreased levels of NADPH and is associated with hemolytic anemia in response to oxidative stress. The red cells of G6PDH deficient persons are susceptible to hemolysis by dietary substances, and by drugs such as primaquine, sulfones, sulfonamides, nitrofurans, vitamin K analogs, acetophenetidin, chloramphenicol, and many others.
Genetic Polymorphisms in Drug Metabolizing Enzymes and Pharmacogenomics
Genetic, environmental, and physiological factors are involved in the regulation of drug biotransformation reactions. Results obtained from epidemiological studies and experimental animal model systems have shown a wide range of phenotypic variation in the ability of individuals to metabolize drugs and environmental chemicals. While some of this variation can be attributed to different environmental exposures, it has become clear that genetic factors also play an important role in determining the response of the individual to exogenous agents. Certain allelic forms of drug-metabolizing enzymes can render the individual either more sensitive or resistant to the toxic or therapeutic effects of exogenous drugs and chemicals. Genetic factors seem to be the major determinants of the variability of drug effects and are responsible for a number of striking quantitative and qualitative differences in pharmacological activity. Genetic differences in the ability of individuals to metabolize a drug through a given pathway are an important contributor to the large interindividual differences of drug efficacy and adverse effects within a population. There are many diverse examples of xenobiotics whose toxicity is directly dependent on the activity of drug-metabolizing enzymes. Often impaired metabolism of a drug through a genetically polymorphic pathway has been associated with an increased incidence of adverse effects in the slow metabolizer population (Weber W. W., Pharmacogenetics, Oxford University Press, N.Y., 1997). Moreover, genetic differences in the regulation, expression and activity of genes coding for Phase I and Phase II drug-metabolizing enzymes can be crucial factors in defining cancer susceptibility and the toxic or carcinogenic power of environmental chemicals and xenobiotics. In addition, the majority of serious cases of drug-drug interactions are a result of the interference of the metabolic clearance of one drug by a coadministered drug. The interference usually occurs via inhibition or induction of drug-metabolizing enzymes. Interindividual differences in susceptibility to severe drug-drug interactions also involve drug-metabolizing enzyme polymorphism. In some cases the design of the drug takes into account the activity of drug-metabolizing enzymes. For example, prodrugs require activation by drug-metabolizing enzymes to exhibit their therapeutic activity. The activation and efficiency of such prodrugs depends on interindividual polymorphism in drug-metabolizing enzymes.
Individual differences in metabolism of therapeutics can lead to severe toxicity or therapeutic failure. Therapeutic management and drug development can be markedly improved by the identification of specific genetic polymorphisms that determine and predict patient susceptibility to diseases or patient responses to drugs. Assessing individual risk rather than population risk will lead to better targeted therapeutic strategies defining individual drug usage based on a benefit/risk prognosis. To assess the origins of individual variations in disease susceptibility or drug response, pharmacogenomics uses the genomic technologies to identify polymorphisms within genes, which are part of biological pathways involved in disease susceptibility, etiology, and development, or more specifically in drug response pathways responsible for a drug""s efficacy, tolerance or toxicity. It can provide tools to refine the design of drug development by decreasing the incidence of adverse events in drug tolerance studies, by better defining patient subpopulations of responders and non-responders in efficacy studies and, by combining the results obtained therefrom, to further allow better enlightened individualized drug usage based on efficacy/tolerance prognosis. Pharmacogenomics can also provide tools to identify new targets for designing drugs and to optimize the use of already existing drugs, in order to either increase their response rate and/or exclude non-responders from corresponding treatment, or decrease their undesirable side effects and/or exclude from corresponding treatment patients with marked susceptibility to undesirable side effects.
Drug-metabolizing enzymes are highly relevant to pharmacogenomics because they are at the core of drug response, drug efficacy and toxicity. Drug-metabolizing enzymes also determine an individual""s susceptibility to exogenous chemicals and to a number of diseases associated with exposure to toxic or carcinogenic chemicals.
The complexity of the pathways and enzymes that are involved in detoxification and metabolism of drugs has limited the precise identification of the drug-metabolizing enzymes, which play the causal role in pathologies or in drug response. Therapeutic management and drug development can be markedly improved by the identification of genetic markers derived from drug-metabolizing enzymes that predict patient susceptibility to diseases or patient responses to drugs.
Genetic Analysis of Complex Traits
Until recently, the identification of genes linked with detectable traits has relied mainly on a statistical approach called linkage analysis. Linkage analysis is based upon establishing a correlation between the transmission of genetic markers and that of a specific trait throughout generations within a family. Linkage analysis involves the study of families with multiple affected individuals and is useful in the detection of inherited traits, which are caused by a single gene, or possibly a very small number of genes. Linkage analysis has been successfully applied to map simple genetic traits that show clear Mendelian inheritance patterns and which have a high penetrance (the probability that a person with a given genotype will exhibit a trait). About 100 pathological trait-causing genes have been discovered using linkage analysis over the last 10 years.
But, most traits of medical relevance do not follow simple Mendelian monogenic inheritance and linkage studies have proven difficult when applied to complex genetic traits. Many complex traits such as height, blood pressure or cancer susceptibility have been known to run in families and are at least partially determined by genetic factors. However, the genes or combination of genes that underlie these observable characteristics or traits remain unknown in most cases. Such complex traits are often due to the combined action of multiple genes as well as environmental factors. Because of their low penetrance, such complex traits do not segregate in a clear-cut Mendelian manner as they are passed from one generation to the next. Drug efficacy, response and tolerance/toxicity can also be considered as multifactoral traits involving a genetic component in the same way as complex diseases. Linkage analysis is impractical when the trait under study is drug response due to the lack of availability of familial cases. In fact, the likelihood of having more than one individual in a family being exposed to the same drug at the same time is very low. Linkage analysis cannot be applied to the study of such traits for which no large informative families are available. Attempts to map complex traits have been plagued by inconclusive results, demonstrating the need for more sophisticated genetic tools.
Knowledge of genetic variation in drug-metabolizing enzymes is important for understanding why some people are more susceptible to toxicity, pathology or respond differently to drugs. Ways to identify genetic polymorphism and to analyze how they impact and predict disease susceptibility and response to treatment are needed.
Whereas a number of polymorphisms and rare mutations have been identified in drug-metabolizing enzymes (see Weber W. W., Pharmacogenetics, Oxford University Press, New York, 1997), genetic markers for use in determining which genes contribute to multigenic or quantitative traits and suitable methods for exploiting those markers have not been found and brought to bare on the genes coding for drug-metabolizing enzymes.
The present invention is based on the discovery of a set of novel DME-related biallelic markers. See Table 11(A-B). These markers are located in the coding regions as well as non-coding regions adjacent to genes which are involved in the metabolic conversion of drugs and other xenobiotics. The position of these markers and knowledge of the surrounding sequence has been used to design polynucleotide compositions which are useful in determining the identity of nucleotides at the marker position, as well as more complex association and haplotyping studies which are useful in determining the genetic basis for variability in drug response and adverse reactions to drugs as well as the genetic basis for disease states involving the metabolic conversion of xenobiotics such as drugs. In addition, the compositions and methods of the invention find use in the identification of the targets for the development of pharmaceutical agents and diagnostic methods, as well as the characterization of the differential efficacious responses to and side effects from pharmaceutical agents.
The present invention further stems from the isolation and characterization of the genomic sequence of the MGST-II gene including its regulatory regions and of the complete cDNA sequence encoding the MGST-II enzyme. Oligonucleotide probes and primers hybridizing specifically with a genomic sequence of MGST-II are also part of the invention. A further object of the invention consists of recombinant vectors comprising any of the nucleic acid sequences described in the present invention, and in particular of recombinant vectors comprising the promoter region of MGST-II or a sequence encoding the MGST-II enzyme, as well as cell hosts comprising said nucleic acid sequences or recombinant vectors. The invention also encompasses methods of screening of molecules which, modulate or inhibit the expression of the MGST-II gene. The invention is also directed to biallelic markers that are located within the MGST-II genomic sequence, these biallelic markers representing useful tools in order to identify a statistically significant association between specific alleles of MGST-II gene and one or several disorders related to asthma and/or hepatotoxicity.
A first embodiment of the invention encompasses polynucleotides consisting of, consisting essentially of, or comprising a contiguous span of nucleotides of a sequence selected as an individual or in any combination from the group consisting of SEQ ID Nos. 1-38, 40-54, 56-353, 355-463, and 465-487, and the complements thereof; preferably SEQ ID Nos. 485-487, 494-531, 533-547, 549-846, 848-956, and 958-977, and the complements thereof; or the sequences described in any one or more of Tables 12, 13, 14, 15, 16, 17, and 18, and the complements thereof, wherein said contiguous span is at least 6, 8, 10, 12, 15, 20, 25, 30, 35, 40, 50, 75, 100, 200, 500, or 1000 nucleotides in length, to the extent that such a length is consistent with the lengths of the particular Sequence ID. The present invention also relates to polynucleotides hybridizing under stringent or intermediate conditions to a sequence selected from the group consisting of SEQ ID Nos. SEQ ID Nos. 1-38, 40-54, 56-353, 355-463, and 465-487, and the complements thereof; preferably SEQ ID Nos. 485-487, 494-531, 533-547, 549-846, 848-956, and 958-977, and the complements thereof. In addition, the polynucleotides of the invention encompass polynucleotides with any further limitation described in this disclosure, or those following, specified alone or in any combination: Said contiguous span may optionally include the DME-related biallelic marker in said sequence; Optionally either the original or the alternative allele of Table 13 may be specified as being present at said DME-related biallelic marker; Optionally either the first or the second allele of Table 12 or 14 may be specified as being present at said DME-related biallelic marker; Optionally, said polynucleotide may comprise, consists of, or consist essentially of a contiguous span which ranges in length from 8, 10, 12, 15, 18 or 20 to 25, 35, 40, 50, 60, 70, or 80 nucleotides, or be specified as being 12, 15, 18, 20, 25, 35, 40, or 50 nucleotides in length and including a DME-related biallelic marker of said sequence, and optionally the original allele of Table 13 is present at said biallelic marker; Optionally, said biallelic marker may be within 6, 5, 4, 3, 2, or 1 nucleotides of the center of said polynucleotide or at the center of said polynucleotide; Optionally, the 3xe2x80x2 end of said contiguous span may be present at the 3xe2x80x2 end of said polynucleotide; Optionally, biallelic marker may be present at the 3xe2x80x2 end of said polynucleotide; Optionally, the 3xe2x80x2 end of said polynucleotide may be located within or at least 2, 4, 6, 8, 10, 12, 15, 18, 20, 25, 50, 100, 250, 500, or 1000 nucleotides upstream of a DME-related biallelic marker in said sequence, to the extent that such a distance is consistent with the lengths of the particular Sequence ID; Optionally, the 3xe2x80x2 end of said polynucleotide may be located 1 nucleotide upstream of a DME-related biallelic marker in said sequence; and Optionally, said polynucleotide may further comprise a label.
A second embodiment of the invention encompasses any polynucleotide of the invention attached to a solid support. In addition, the polynucleotides of the invention which are attached to a solid support encompass polynucleotides with any further limitation described in this disclosure, or those following, specified alone or in any combination: Optionally, said polynucleotides may be specified as attached individually or in groups of at least 2, 5, 8, 10, 12, 15, 20, or 25 distinct polynucleotides of the inventions to a single solid support; Optionally, polynucleotides other than those of the invention may attached to the same solid support as polynucleotides of the invention; Optionally, when multiple polynucleotides are attached to a solid support they may be attached at random locations, or in an ordered array; Optionally, said ordered array may be addressable.
A third embodiment of the invention encompasses the use of any polynucleotide for, or any polynucleotide for use in, determining the identity of one or more nucleotides at a DME-related biallelic marker. In addition, the polynucleotides of the invention for use in determining the identity of one or more nucleotides at a DME-related biallelic marker encompass polynucleotides with any further limitation described in this disclosure, or those following, specified alone or in any combination. Optionally, said DME-related biallelic marker may be in a sequence selected individually or in any combination from the group consisting of SEQ ID Nos. 1-38, 40-54, 56-353, 355-463, and 465-487, and the complements thereof; preferably SEQ ID Nos. 485-487, 494-531, 533-547, 549-846, 848-956, and 958-977, and the complements thereof; Optionally, said polynucleotide may comprise a sequence disclosed in the present specification; Optionally, said polynucleotide may consist of, or consist essentially of any polynucleotide described in the present specification; Optionally, said determining may be performed in a hybridization assay, sequencing assay, microsequencing assay, or an enzyme-based mismatch detection assay; Optionally, said polynucleotide may be attached to a solid support, array, or addressable array; Optionally, said polynucleotide may be labeled.
A fourth embodiment of the invention encompasses the use of any polynucleotide for, or any polynucleotide for use in, amplifying a segment of nucleotides comprising a DME-related biallelic marker. In addition, the polynucleotides of the invention for use in amplifying a segment of nucleotides comprising a DME-related biallelic marker encompass polynucleotides with any further limitation described in this disclosure, or those following, specified alone or in any combination: Optionally, said DME-related biallelic marker may be in a sequence selected individually or in any combination from the group consisting of SEQ ID Nos. 1-38, 40-54, 56-353, 355-463, and 465-487, and the complements thereof; preferably SEQ ID Nos. 485-487, 494-531, 533-547, 549-846, 848-956, and 958-977, and the complements thereof; Optionally, said DME-related biallelic marker may be selected individually or in any combination from the biallelic markers described in Table 11(A-B); Optionally, said DME-related biallelic marker may be selected from the biallelic markers found in Tables 19, 20, 21 and 22; Optionally, said DME-related biallelic marker may be selected from the following biallelic markers: 12-455-326, 12-453-429, 12-454-363, 12-441-233, 12-461-299, 12-426-154, 12-424-198, 12-716-295, 10-428-219, 12-720-80, 12-156-91, 12-140-134, 12-653-423, 10-471-84, 10-471-85, 10-470-25, 12-652-203, 12-637-219, 12-721-440, and 10-420-284; Optionally, said polynucleotide may comprise a sequence disclosed in the present specification; Optionally, said polynucleotide may consist of, or consist essentially of any polynucleotide described in the present specification; Optionally, said amplifying may be performed by a PCR or LCR. Optionally, said polynucleotide may be attached to a solid support, array, or addressable array. Optionally, said polynucleotide may be labeled.
A fifth embodiment of the invention encompasses methods of genotyping a biological sample comprising determining the identity of a nucleotide at a DME-related biallelic marker. In addition, the genotyping methods of the invention encompass methods with any further limitation described in this disclosure, or those following, specified alone or in any combination: Optionally, said DME-related biallelic marker may be in a sequence selected individually or in any combination from the group consisting of SEQ ID Nos. 1-38, 40-54, 56-353, 355-463, and 465-487, and the complements thereof; preferably SEQ ID Nos. 485-487, 494-531, 533-547, 549-846, 848-956, and 958-977, and the complements thereof; Optionally, said DME-related biallelic marker may be selected individually or in any combination from the biallelic markers described in Table 11(A-B); Optionally, said DME-related biallelic marker may be selected individually or in any combination from the biallelic markers described in Table 11(A-B); Optionally, said DME-related biallelic marker may be selected from the biallelic markers found in Tables 19, 20, 21 and 22; Optionally, said DME-related biallelic marker may be selected from the following biallelic markers: 12-455-326, 12-453-429, 12-454-363, 12-441-233, 12-461-299, 12-426-154, 12-424-198, 12-716-295, 10-428-219, 12-720-80, 12-156-91, 12-140-134, 12-653-423, 10-471-84, 10-471-85, 10-470-25, 12-652-203, 12-637-219, 12-721-440, and 10-420-284; Optionally, said method further comprises determining the identity of a second nucleotide at said biallelic marker, wherein said first nucleotide and second nucleotide are not base paired (by Watson and Crick base pairing) to one another; Optionally, said biological sample is derived from a single individual or subject; Optionally, said method is performed in vitro; Optionally, said biallelic marker is determined for both copies of said biallelic marker present in said individual""s genome; Optionally, said biological sample is derived from multiple subjects or individuals; Optionally, said method further comprises amplifying a portion of said sequence comprising the biallelic marker prior to said determining step; Optionally, wherein said amplifying is performed by PCR, LCR, or replication of a recombinant vector comprising an origin of replication and said portion in a host cell; Optionally, wherein said determining is performed by a hybridization assay, sequencing assay, microsequencing assay, or an enzyme-based mismatch detection assay.
A sixth embodiment of the invention comprises methods of estimating the frequency of an allele in a population comprising genotyping individuals from said population for a DME-related biallelic marker and determining the proportional representation of said biallelic marker in said population. In addition, the methods of estimating the frequency of an allele in a population of the invention encompass methods with any further limitation described in this disclosure, or those following, specified alone or in any combination: Optionally, said DME-related biallelic marker may be in a sequence selected individually or in any combination from the group consisting of SEQ ID Nos. 1-38, 40-54, 56-353, 355-463, and 465-487, and the complements thereof; preferably SEQ ID Nos. 485-487, 494-531, 533-547, 549-846, 848-956, and 958-977, and the complements thereof; Optionally, said DME-related biallelic marker may be selected from the biallelic markers described in Table 11(A-B); Optionally, said DME-related biallelic marker may be selected individually or in any combination from the biallelic markers described in Table 11(A-B); Optionally, said DME-related biallelic marker may be selected from the biallelic markers found in Tables 19, 20, 21 and 22; Optionally, said DME-related biallelic marker may be selected from the following biallelic markers: 12-455-326, 12-453-429, 12-454-363, 12-441-233, 12-461-299, 12-426-154, 12-424-198, 12-716-295, 10-428-219, 12-720-80, 12-156-91, 12-140-134, 12-653-423, 10-471-84, 10-471-85, 10-470-25, 12-652-203, 12-637-219, 12-721-440, and 10-420-284; Optionally, determining the frequency of a biallelic marker allele in a population may be accomplished by determining the identity of the nucleotides for both copies of said biallelic marker present in the genome of each individual in said population and calculating the proportional representation of said nucleotide at said DME-related biallelic marker for the population; Optionally, determining the frequency of a biallelic marker allele in a population may be accomplished by performing a genotyping method on a pooled biological sample derived from a representative number of individuals, or each individual, in said population, and calculating the proportional amount of said nucleotide compared with the total.
A seventh embodiment of the invention comprises methods of detecting an association between an allele and a phenotype, comprising the steps of a) determining the frequency of at least one DME-related biallelic marker allele in a case (trait positive) population, b) determining the frequency of said DME-related biallelic marker allele in a control population and; c) determining whether a statistically significant association exists between said genotype and said phenotype. In addition, the methods of detecting an association between an allele and a phenotype of the invention encompass methods with any further limitation described in this disclosure, or those following, specified alone or in any combination: Optionally, said DME-related biallelic marker may be in a sequence selected individually or in any combination from the group consisting of SEQ ID Nos. 1-38, 40-54, 56-353, 355-463, and 465-487, and the complements thereof; preferably SEQ ID Nos. 485-487, 494-531, 533-547, 549-846, 848-956, and 958-977, and the complements thereof; Optionally, said DME-related biallelic marker may be selected from the biallelic markers described in Table 11(A-B); Optionally, said control population may be a trait negative population, or a random population; Optionally, said phenotype is a response to a drug, or a side effects to a drug, or a disease involving the metabolic conversion of xenobiotics; Optionally, the identity of the nucleotides at the biallelic markers in everyone of the following sequences: SEQ ID Nos. 1-38, 40-54, 56-353, 355-463, and 465-487; preferably SEQ ID Nos. 485-487, 494-531, 533-547, 549-846, 848-956, and 958-977 is determined in steps a) and b).
An eighth embodiment of the present invention encompasses methods of estimating the frequency of a haplotype for a set of biallelic markers in a population, comprising the steps of: a) genotyping each individual in said population for at least one DME-related biallelic marker, b) genotyping each individual in said population for a second biallelic marker by determining the identity of the nucleotides at said second biallelic marker for both copies of said second biallelic marker present in the genome; and c) applying a haplotype determination method to the identities of the nucleotides determined in steps a) and b) to obtain an estimate of said frequency. In addition, the methods of estimating the frequency of a haplotype of the invention encompass methods with any further limitation described in this disclosure, or those following, specified alone or in any combination: Optionally said haplotype determination method is selected from the group consisting of asymmetric PCR amplification, double PCR amplification of specific alleles, the Clark method, and an expectation maximization algorithm; Optionally, said second biallelic marker is a DME-related biallelic marker in a sequence selected from the group consisting of the biallelic markers of SEQ ID Nos. 1-38, 40-54, 56-353, 355-463, and 465-487, and the complements thereof; preferably SEQ ID Nos. 485-487, 494-531, 533-547, 549-846, 848-956, and 958-977, and the complements thereof; Optionally, said DME-related biallelic markers may be selected individually or in any combination from the biallelic markers described in Table 11(A-B); Optionally, said DME-related biallelic marker may be selected individually or in any combination from the biallelic markers described in Table 11(A-B); Optionally, said DME-related biallelic marker may be selected from the biallelic markers found in Tables 19, 20, 21 and 22; Optionally, said DME-related biallelic marker may be selected from the following biallelic markers: 12-455-326, 12-453-429, 12-454-363, 12-441-233, 12-461-299, 12-426-154, 12-424-198, 12-716-295, 10-428-219, 12-720-80, 12-156-91, 12-140-134, 12-653-423, 10-471-84, 10-471-85, 10-470-25, 12-652-203, 12-637-219, 12-721-440, and 10-420-284; Optionally, the identity of the nucleotides at the biallelic markers in everyone of the sequences of SEQ ID Nos. 1-38, 40-54, 56-353, 355-463, and 465-487; preferably SEQ ID Nos. 485-487, 494-531, 533-547, 549-846, 848-956, and 958-977 is determined in steps a) and b).
A ninth embodiment of the present invention encompasses methods of detecting an association between a haplotype and a phenotype, comprising the steps of: a) estimating the frequency of at least one haplotype in a trait positive population according to a method of estimating the frequency of a haplotype of the invention; b) estimating the frequency of said haplotype in a control population according to the method of estimating the frequency of a haplotype of the invention; and c) determining whether a statistically significant association exists between said haplotype and said phenotype. In addition, the methods of detecting an association between a haplotype and a phenotype of the invention encompass methods with any further limitation described in this disclosure, or those following, specified alone or in any combination: Optionally, said DME-related biallelic marker may be in a sequence selected individually or in any combination from the group consisting of SEQ ID Nos. 1-38, 40-54, 56-353, 355-463, and 465-487, and the complements thereof; preferably SEQ ID Nos. 485-487, 494-531, 533-547, 549-846, 848-956, and 958-977, and the complements thereof; Optionally, said DME-related biallelic markers may be selected individually or in any combination from the biallelic markers described in Table 11(A-B); Optionally, said DME-related biallelic marker may be selected from the biallelic markers found in Tables 19, 20, 21 and 22; Optionally, said DME-related biallelic marker may be selected from the following biallelic markers: 12-455-326, 12-453-429, 12-454-363, 12-441-233, 12-461-299, 12-426-154, 12-424-198, 12-716-295, 10-428-219, 12-720-80, 12-156-91, 12-140-134, 12-653-423, 10-471-84, 10-471-85, 10-470-25, 12-652-203, 12-637-219, 12-721-440, and 10-420-284; Optionally, said control population may be a trait negative population, or a random population; Optionally, said phenotype is a response to a drug, or a side effects to a drug, or a disease involving the metabolic conversion of xenobiotics; Optionally, the identity of the nucleotides at the biallelic markers in everyone of the following sequences: SEQ ID Nos. 1-38, 40-54, 56-353, 355-463, and 465-487; preferably SEQ ID Nos. 485-487, 494-531, 533-547, 549-846, 848-956, and 958-977 is determined in steps a) and b).
A tenth embodiment of the present invention is a method of administering a drug or a treatment comprising the steps of: a) obtaining a nucleic acid sample from an individual; b) determining the identity of the polymorphic base of at least one DME-related biallelic marker which is associated with a positive response to the treatment or the drug; or at least one biallelic DME-related biallelic marker which is associated with a negative response to the treatment or the drug; and c) administering the treatment or the drug to the individual if the nucleic acid sample contains said biallelic marker associated with a positive response to the treatment or the drug or if the nucleic acid sample lacks said biallelic marker associated with a negative response to the treatment or the drug. In addition, the methods of the present invention for administering a drug or a treatment encompass methods with any further limitation described in this disclosure, or those following, specified alone or in any combination: optionally, said DME-related biallelic marker may be in a sequence selected individually or in any combination from the group consisting of SEQ ID Nos. 1-38, 40-54, 56-353, 355-463, and 465-487, and the complements thereof; or preferably SEQ ID Nos. 485-487, 494-531, 533-547, 549-846, 848-956, 958-977, and the complements thereof; or more preferably SEQ ID Nos. 496, 498, 502, 506-508, 518, 524, 526, 530, 531, 534, 816, 838, 844-846, 850, 870, 873, and the complements thereof; Optionally, the administering step comprises administering the drug or the treatment to the individual if the nucleic acid sample contains said biallelic marker associated with a positive response to the treatment or the drug and the nucleic acid sample lacks said biallelic marker associated with a negative response to the treatment or the drug.
An eleventh embodiment of the present invention is a method of selecting an individual for inclusion in a clinical trial of a treatment or drug comprising the steps of: a) obtaining a nucleic acid sample from an individual; b) determining the identity of the polymorphic base of at least one DME-related biallelic marker which is associated with a positive response to the treatment or the drug, or at least one DME-related biallelic marker which is associated with a negative response to the treatment or the drug in the nucleic acid sample, and c) including the individual in the clinical trial if the nucleic acid sample contains said DME-related biallelic marker associated with a positive response to the treatment or the drug or if the nucleic acid sample lacks said biallelic marker associated with a negative response to the treatment or the drug. In addition, the methods of the present invention for selecting an individual for inclusion in a clinical trial of a treatment or drug encompass methods with any further limitation described in this disclosure, or those following, specified alone or in any combination: Optionally, said DME-related biallelic marker may be in a sequence selected individually or in any combination from the group consisting of SEQ ID Nos. 1-38, 40-54, 56-353, 355-463, and 465-487, and the complements thereof, or preferably SEQ ID Nos. 485-487, 494-531, 533-547, 549-846, 848-956, 958-977, and the complements thereof; or more preferably SEQ ID Nos. 496, 498, 502, 506-508, 518, 524, 526, 530, 531, 534, 816, 838, 844-846, 850, 870, 873, and the complements thereof; Optionally, the including step comprises administering the drug or the treatment to the individual if the nucleic acid sample contains said biallelic marker associated with a positive response to the treatment or the drug and the nucleic acid sample lacks said biallelic marker associated with a negative response to the treatment or the drug.
Additional embodiments are set forth in the Detailed Description of the Invention and in the Examples.
Table 11A is a chart containing a list of all of the DME-related biallelic markers for each gene with an indication of the gene for which the marker is in closest physical proximity, an indication of whether the markers have been validated by microsequencing (with a Y indicating that the markers have been validated by microsequencing and an N indicating that it has not), and an indication of the identity and frequency of the least common allele determined by genotyping (with a blank left to indicate that the frequency has not yet been reported for some markers). The frequencies were determined from DNA samples collected from a random US Caucasian population. When the marker was determined to be homozygous at the particular location for the random US Caucasian population, the homozygous bases were recorded in the xe2x80x9cGenotyping Least Common Allele Frequencyxe2x80x9d column of Table 11A.
Table 11B contains all of the DME-related biallelic markers provided in Table 11A; however, they are provided in shorter, easier to search sequences of 47 nucleotides. Accordingly, Table 11A begins with SEQ ID No. 1 and ends with SEQ ID No. 484, while Table 11B begins with SEQ ID No. 494 and ends with SEQ ID No. 977 (SEQ ID Nos. 485-493 correspond to the genomic and protein sequences of the invention and are not repeated in Table 11B). Table 1 below contains the first five markers listed in the sequence listing and their corresponding SEQ ID numbers in Tables 11A and 11B to illustrate the relationship between Tables 11A and 11B:
Table 11B is the same as Table 11A in that it is a list of all of the DME-related biallelic markers for each gene with an indication of the gene for which the marker is in closest physical proximity, an indication of whether the markers have been validated by microsequencing (with a Y indicating that the markers have been validated by microsequencing and an N indicating that it has not), and an indication of the identity and frequency of the least common allele determined by genotyping (with a blank left to indicate that the frequency has not yet been reported for some markers). However, the xe2x80x9cBiallelic Marker Position in SEQ ID No.xe2x80x9d for all of the DME-related biallelic markers provided in Table 11B is position 24 (representing the midpoint of the 47 mers that make up Table 11B). The frequencies were determined from DNA samples collected from a random US Caucasian population.
Tables 12, 13, and 14 are charts containing lists of the DME-related biallelic markers. Each marker is described by indicating its SEQ ID, the biallelic marker ID, and the two most common alleles. Table 12 is a chart containing a list of biallelic markers surrounded by preferred sequences. In the column labeled, xe2x80x9cPOSITION RANGE OF PREFERRED SEQUENCExe2x80x9d of Table 12, regions of particularly preferred sequences are listed for each SEQ ID, which contain a DME-related biallelic marker, as well as particularly preferred regions of sequences that may not contain a DME-related biallelic marker but, which are in sufficiently close proximity to a DME-related biallelic marker to be useful as amplification or sequencing primers.
Table 15 is a chart listing particular preferred sequences that are useful for designing some of the primers and probes of the invention. Each sequence is described by indicating its Sequence ID and the positions of the first and last nucleotides (position range) of the particular sequence in the Sequence ID.
Table 16 is a chart listing microsequencing primers which have been used to genotype DME-related biallelic markers (indicated by an *) and other preferred microsequencing primers for use in genotyping DME-related biallelic markers. Each of the primers which falls within the strand of nucleotides included in the Sequence Listing are described by indicating their Sequence ID number and the positions of the first and last nucleotides (position range) of the primers in the Sequence ID. Since the sequences in the Sequence Listing are single stranded and half the possible microsequencing primers are composed of nucleotide sequences from the complementary strand, the primers that are composed of nucleotides in the complementary strand are described by indicating their SEQ ID numbers and the positions of the first and last nucleotides to which they are complementary (complementary position range) in the Sequence ID.
Table 17 is a chart listing amplification primers which have been used to amplify polynucleotides containing one or more DME-related biallelic markers. Each of the primers which falls within the strand of nucleotides included in the Sequence Listing are described by indicating their Sequence ID number and the positions of the first and last nucleotides (position range) of the primers in the Sequence ID. Since the sequences in the Sequence Listing are single stranded and half the possible amplification primers are composed of nucleotide sequences from the complementary strand, the primers that are composed of nucleotides in the complementary strand are defined by the SEQ ID numbers and the positions of the first and last nucleotides to which they are complementary (complementary position range) in the Sequence ID.
Table 18 is a chart listing preferred probes useful in genotyping DME-related biallelic markers by hybridization assays. The probes are generally 25-mers with a DME-related biallelic marker in the center position, and described by indicating their Sequence ID number and the positions of the first and last nucleotides (position range) of the probes in the Sequence ID. The probes complementary to the sequences in each position range in each Sequence ID are also understood to be a part of this preferred list even though they are not specified separately.
Table 19 is a chart containing a list of preferred MGST-II-related biallelic markers with an indication of the frequency of both alleles determined by genotyping. Frequencies were determined in a random US Caucasian population.
Table 20 is a chart containing a list of preferred ME1-related biallelic markers with an indication of the frequency of both alleles determined by genotyping. Frequencies were determined in a random US Caucasian population.
Table 21 is a chart containing a list of preferred UGT1A7-related biallelic markers with an indication of the frequency of both alleles determined by genotyping. Frequencies were determined in a random US Caucasian population and a random French Caucasian population.
Table 22 is a chart containing a list of preferred UGT2B4-related biallelic markers with an indication of the frequency of both alleles determined by genotyping. Frequencies were determined in a random US Caucasian population and a random French Caucasian population.
Table 23 is a chart showing the results of a haplotype analysis study demonstrating an association between asthma and MGST-II-related biallelic marker haplotypes.
Table 24 is a chart showing the results of a permutation test which evaluates the statistical significance of the results obtained for the MGST2 haplotype analysis.
Table 25 is a chart showing the results of a single point allelic association analysis conducted on individuals with side effects to treatment with the anti-asthmatic drug Zyflo(trademark) (zileuton) and MGST-II related biallelic markers.
Table 26 is a chart showing the results of a haplotype analysis study demonstrating an association between side effects upon treatment with the anti-asthmatic drug Zyflo(trademark) (zileuton) and MGST-II related biallelic marker haplotypes.
Table 27 is a table showing the results of a phenotypic permutation test which evaluates the statistical significance of the MGST-II haplotype analysis results.
Table 28 is a chart showing the results of a single point allelic association analysis conducted on individuals with side effects to treatment with the anti-asthmatic drug Zyflo(trademark) (zileuton) and UGT1A7-related biallelic markers.
Table 29 is a chart showing the results of a haplotype analysis study demonstrating an association between side effects upon treatment with the anti-asthmatic drug Zyflo(trademark) (zileuton) and UGT1A7-related biallelic marker haplotypes.
Table 30 is a chart showing the results of a permutation test which evaluates the statistical significance of the results obtained from the UGT1A7 haplotype analysis.
Table 31 is a chart showing the results of a single point allelic association analysis conducted on individuals with side effects to treatment with the anti-asthmatic drug Zyflo(trademark) (zileuton) and UGT2B4-related biallelic markers.
Table 32 is a chart showing the results of a haplotype analysis study demonstrating an association between side effects upon treatment with the anti-asthmatic drug Zyflo(trademark) (zileuton) and UGT2B4-related biallelic marker haplotypes.
Table 33 is a chart showing the results of a permutation test which evaluates the statistical significance of the results obtained from the UGT2B4 haplotype analysis.
SEQ ID Nos. 1-484 contain the nucleotide sequence of genomic DNA fragments located in the vicinity of the candidate genes.
SEQ ID No. 485 contains the genomic sequence of the MGST-II gene comprising the 5xe2x80x2 regulatory region (upstream untranscribed region), the exons and introns, and the 3xe2x80x2 regulatory region (downstream untranscribed region).
SEQ ID No. 486 contains a cDNA sequence of MGST-II.
SEQ ID No. 487 contains a cDNA sequence of MGST-II corresponding to an alternative messenger RNA which is due to alternative splicing joining exon 1 to exon 3 and resulting in the absence of exon 2.
SEQ ID No. 488 contains the amino acid sequence encoded by the cDNA of SEQ ID No. 486.
SEQ ID No. 489 contains the amino acid sequence encoded by the cDNA of SEQ ID No. 487.
SEQ ID No. 490 contains a partial cDNA sequence of MGST-II sequence corresponding to a cloned partial messenger RNA.
SEQ ID No. 491 contains a partial cDNA sequence of MGST-II sequence corresponding to a cloned partial messenger RNA.
SEQ ID No. 492 contains a primer containing the additional PU 5xe2x80x2 sequence described further in Example 1.
SEQ ID No. 493 contains a primer containing the additional RP 5xe2x80x2 sequence described further in Example 1.
SEQ ID Nos. 494-977 contain the nucleotide sequence of genomic DNA fragments located in the vicinity of the candidate genes. These sequences are in easier to search sequences of 47 nucleotides.
Advantages of the Biallelic Markers of the Present Invention
The DME-related biallelic markers of the present invention offer a number of important advantages over other genetic markers such as RFLP (Restriction fragment length polymorphism) and VNTR (Variable Number of Tandem Repeats) markers.
The first generation of markers, were RFLPs, which are variations that modify the length of a restriction fragment. But methods used to identify and to type RFLPs are relatively wasteful of materials, effort, and time. The second generation of genetic markers were VNTRs, which can be categorized as either minisatellites or microsatellites. Minisatellites are tandemly repeated DNA sequences present in units of 5-50 repeats which are distributed along regions of the human chromosomes ranging from 0.1 to 20 kilobases in length. Since they present many possible alleles, their informative content is very high. Minisatellites are scored by performing Southern blots to identify the number of tandem repeats present in a nucleic acid sample from the individual being tested. However, there are only 104 potential VNTRs that can be typed by Southern blotting. Moreover, both RFLP and VNTR markers are costly and time-consuming to develop and assay in large numbers.
Single nucleotide polymorphism or biallelic markers can be used in the same manner as RFLPs and VNTRs but offer several advantages. Single nucleotide polymorphisms are densely spaced in the human genome and represent the most frequent type of variation. An estimated number of more than 107 sites are scattered along the 3xc3x97109 base pairs of the human genome. Therefore, single nucleotide polymorphism occur at a greater frequency and with greater uniformity than RFLP or VNTR markers which means that there is a greater probability that such a marker will be found in close proximity to a genetic locus of interest. Single nucleotide polymorphisms are less variable than VNTR markers but are mutationally more stable.
Also, the different forms of a characterized single nucleotide polymorphism, such as the biallelic markers of the present invention, are often easier to distinguish and can therefore be typed easily on a routine basis. Biallelic markers have single nucleotide based alleles and they have only two common alleles, which allows highly parallel detection and automated scoring. The biallelic markers of the present invention offer the possibility of rapid, high-throughput genotyping of a large number of individuals.
Biallelic markers are densely spaced in the genome, sufficiently informative and can be assayed in large numbers. The combined effects of these advantages make biallelic markers extremely valuable in genetic studies. Biallelic markers can be used in linkage studies in families, in allele sharing methods, in linkage disequilibrium studies in populations, in association studies of case-control populations. An important aspect of the present invention is that biallelic markers allow association studies to be performed to identify genes involved in complex traits. Association studies examine the frequency of marker alleles in unrelated case-and control-populations and are generally employed in the detection of polygenic or sporadic traits. Association studies may be conducted within the general population and are not limited to studies performed on related individuals in affected families (linkage studies). Biallelic markers in different genes can be screened in parallel for direct association with disease or response to a treatment. This multiple gene approach is a powerful tool for a variety of human genetic studies as it provides the necessary statistical power to examine the synergistic effect of multiple genetic factors on a particular phenotype, drug response, sporadic trait, or disease state with a complex genetic etiology.
Candidate Genes of the Present Invention
Different approaches can be employed to perform association studies: genome-wide association studies, candidate region association studies and candidate gene association studies. Genome-wide association studies rely on the screening of genetic markers evenly spaced and covering the entire genome. Candidate region association studies rely on the screening of genetic markers evenly spaced covering a region identified as linked to the trait of interest. The candidate gene approach is based on the study of genetic markers specifically derived from genes potentially involved in a biological pathway related to the trait of interest. In the present invention, genes involved in drug metabolism have been chosen as candidate genes. As mentioned above, these genes are highly relevant to pharmacogenetics because they are at the core of drug response, drug efficacy and toxicity, moreover, drug-metabolizing enzymes also determine an individuals susceptibility to exogenous chemicals and to a number of diseases associated with exposure to toxic or carcinogenic chemicals. The candidate gene analysis clearly provides a short-cut approach to the identification of genes and gene polymorphisms related to a particular trait when some information concerning the biology of the trait is available. However, it should be noted that all of the biallelic markers disclosed in the instant application can be employed as part of genome-wide association studies or as part of candidate region association studies and such uses are specifically contemplated in the present invention and claims. All of the markers are known to be in close proximity to the genes with which they are listed in Table 11(A-B). For a portion of the markers, the precise position of the marker with respect to the various coding and non-coding elements of the genes has also been determined.
The following is a table of abbreviations for the candidate genes as they appear throughout the specification:
Definitions
As used interchangeably herein, the terms xe2x80x9cnucleic acids,xe2x80x9d xe2x80x9coligonucleotidesxe2x80x9d and xe2x80x9cpolynucleotidesxe2x80x9d include RNA, DNA, or RNA/DNA hybrid sequences of more than one nucleotide in either single chain or duplex form. The term xe2x80x9cnucleotidexe2x80x9d as used herein as an adjective to describe molecules comprising RNA, DNA, or RNA/DNA hybrid sequences of any length in single-stranded or duplex form. The term xe2x80x9cnucleotidexe2x80x9d is also used herein as a noun to refer to individual nucleotides or varieties of nucleotides, meaning a molecule, or individual unit in a larger nucleic acid molecule, comprising a purine or pyrimidine, a ribose or deoxyribose sugar moiety, and a phosphate group, or phosphodiester linkage in the case of nucleotides within an oligonucleotide or polynucleotide. Although the term xe2x80x9cnucleotidexe2x80x9d is also used herein to encompass xe2x80x9cmodified nucleotidesxe2x80x9d which comprise at least one modifications (a) an alternative linking group, (b) an analogous form of purine, (c) an analogous form of pyrimidine, or (d) an analogous sugar, for examples of analogous linking groups, purine, pyrimidines, and sugars see for example PCT publication No. WO 95/04064, the disclosure of which is incorporated herein by reference in its entirety. However, the polynucleotides of the invention are preferably comprised of greater than 50% conventional deoxyribose nucleotides, and most preferably greater than 90% conventional deoxyribose nucleotides. The polynucleotide sequences of the invention may be prepared by any known method, including synthetic, recombinant, ex vivo generation, or a combination thereof, as well as utilizing any purification methods known in the art.
Throughout the present specification, the expression xe2x80x9cnucleotide sequencexe2x80x9d may be employed to designate indifferently a polynucleotide or a nucleic acid. More precisely, the expression xe2x80x9cnucleotide sequencexe2x80x9d encompasses the nucleic material itself and is thus not restricted to the sequence information (i.e. the succession of letters chosen among the four base letters) that biochemically characterizes a specific DNA or RNA molecule.
The term xe2x80x9cpolypeptidexe2x80x9d refers to a polymer of amino without regard to the length of the polymer; thus, peptides, oligopeptides, and proteins are included within the definition of polypeptide. This term also does not specify or exclude prost-expression modifications of polypeptides, for example, polypeptides which include the covalent attachment of glycosyl groups, acetyl groups, phosphate groups, lipid groups and the like are expressly encompassed by the term polypeptide. Also included within the definition are polypeptides which contain one or more analogs of an amino acid (including, for example, non-naturally occurring amino acids, amino acids which only occur naturally in an unrelated biological system, modified amino acids from mammalian systems etc.), polypeptides with substituted linkages, as well as other modifications known in the art, both naturally occurring and non-naturally occurring.
The term xe2x80x9crecombinant polypeptidexe2x80x9d is used herein to refer to polypeptides that have been artificially designed and which comprise at least two polypeptide sequences that are not found as contiguous polypeptide sequences in their initial natural environment, or to refer to polypeptides which have been expressed from a recombinant polynucleotide.
As used herein, the term xe2x80x9cisolatedxe2x80x9d requires that the material be removed from its original environment (e.g., the natural environment if it is naturally occurring). For example, a naturally occurring polynucleotide present in a living animal is not isolated, but the same polynucleotide, separated from some or all of the coexisting materials in the natural system, is isolated. Specifically excluded from the definition of xe2x80x9cisolatedxe2x80x9d are: naturally occurring chromosomes (e.g., chromosome spreads) artificial chromosome libraries, genomic libraries, and cDNA libraries that exist either as an in vitro nucleic acid preparation or as a transfected/transformed host cell preparation, wherein the host cells are either an in vitro heterogeneous preparation or plated as a heterogeneous population of single colonies. Also specifically excluded are the above libraries wherein the 5xe2x80x2 EST makes up less than 5% of the number of nucleic acid inserts in the vector molecules. Further specifically excluded are whole cell genomic DNA or whole cell RNA preparations (including said whole cell preparations which are mechanically sheared or enzymaticly digested). Further specifically excluded are the above whole cell preparations as either an in vitro preparation or as a heterogeneous mixture separated by electrophoresis (including blot transfers of the same) wherein the polynucleotide of the invention have not been further separated from the heterologous polynucleotides in the electrophoresis medium (e.g., further separating by excising a single band from a heterogeneous band population in an agarose gel or nylon blot).
As used herein, the term xe2x80x9cpurifiedxe2x80x9d does not require absolute purity; rather, it is intended as a relative definition. Individual 5xe2x80x2 EST clones isolated from a cDNA library have been conventionally purified to electrophoretic homogeneity. The sequences obtained from these clones could not be obtained directly either from the library or from total human DNA. The cDNA clones are not naturally occurring as such, but rather are obtained via manipulation of a partially purified naturally occurring substance (messenger RNA). The conversion of mRNA into a cDNA library involves the creation of a synthetic substance (cDNA) and pure individual cDNA clones can be isolated from the synthetic library by clonal selection. Thus, creating a cDNA library from messenger RNA and subsequently isolating individual clones from that library results in an approximately 104-106 fold purification of the native message. Purification of starting material or natural material to at least one order of magnitude, preferably two or three orders, and more preferably four or five orders of magnitude is expressly contemplated. Alternatively, purification may be expressed as xe2x80x9cat leastxe2x80x9d a percent purity relative to heterologous polynucleotides (DNA, RNA or both). As a preferred embodiment, the polynucleotides of the present invention are at least; 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 96%, 98%, 99%, or 100% pure relative to heterologous polynucleotides. As a further preferred embodiment the polynucleotides have an xe2x80x9cat leastxe2x80x9d purity ranging from any number, to the thousandth position, between 90% and 100% (e.g., 5xe2x80x2 EST at least 99.995% pure) relative to heterologous polynucleotides. Additionally, purity of the polynucleotides may be expressed as a percentage (as described above) relative to all materials and compounds other than the carrier solution. Each number, to the thousandth position, may be claimed as individual species of purity.
As used herein, the term xe2x80x9cnon-human animalxe2x80x9d refers to any non-human vertebrate, birds and more usually mammals, preferably primates, farm animals such as swine, goats, sheep, donkeys, and horses, rabbits or rodents, more preferably rats or mice. As used herein, the term xe2x80x9canimalxe2x80x9d is used to refer to any vertebrate, preferable a mammal. Both the terms xe2x80x9canimalxe2x80x9d and xe2x80x9cmammalxe2x80x9d expressly embrace human subjects unless preceded with the term xe2x80x9cnon-humanxe2x80x9d.
The term xe2x80x9cprimerxe2x80x9d denotes a specific oligonucleotide sequence which is complementary to a target nucleotide sequence and used to hybridize to the target nucleotide sequence. A primer serves as an initiation point for nucleotide polymerization catalyzed by either DNA polymerase, RNA polymerase or reverse transcriptase.
The term xe2x80x9cprobexe2x80x9d denotes a defined nucleic acid segment (or nucleotide analog segment, e.g., polynucleotide as defined herein) which can be used to identify a specific polynucleotide sequence present in samples, said nucleic acid segment comprising a nucleotide sequence complementary of the specific polynucleotide sequence to be identified.
The terms xe2x80x9ctraitxe2x80x9d and xe2x80x9cphenotypexe2x80x9d are used interchangeably herein and refer to any visible, detectable or otherwise measurable property of an organism such as symptoms of, or susceptibility to a disease for example. Typically the terms xe2x80x9ctraitxe2x80x9d or xe2x80x9cphenotypexe2x80x9d are used herein to refer to symptoms of, or susceptibility to a disease; or to refer to an individual""s response to a drug; or to refer to symptoms of, or susceptibility to side effects to a drug. In addition, the terms xe2x80x9ctraitxe2x80x9d or xe2x80x9cphenotypexe2x80x9d may be used herein to refer to symptoms of, or susceptibility to a disease involving arachidonic acid metabolism; or to refer to an individual""s response to an agent acting on arachidonic acid metabolism; or to refer to symptoms of, or susceptibility to side effects to an agent acting on arachidonic acid metabolism.
The term xe2x80x9callelexe2x80x9d is used herein to refer to variants of a nucleotide sequence. A biallelic polymorphism has two forms. Typically the first identified allele is designated as the original allele whereas other alleles are designated as alternative alleles. Diploid organisms may be homozygous or heterozygous for an allelic form.
The term xe2x80x9cheterozygosity ratexe2x80x9d is used herein to refer to the incidence of individuals in a population, which are heterozygous at a particular allele. In a biallelic system the heterozygosity rate is on average equal to 2Pa(1xe2x88x92Pa), where Pa is the frequency of the least common allele. In order to be useful in genetic studies a genetic marker should have an adequate level of heterozygosity to allow a reasonable probability that a randomly selected person will be heterozygous.
The term xe2x80x9cgenotypexe2x80x9d as used herein refers the identity of the alleles present in an individual or a sample. In the context of the present invention a genotype preferably refers to the description of the biallelic marker alleles present in an individual or a sample. The term xe2x80x9cgenotypingxe2x80x9d a sample or an individual for a biallelic marker consists of determining the specific allele or the specific nucleotide carried by an individual at a biallelic marker.
The term xe2x80x9cmutationxe2x80x9d as used herein refers to a difference in DNA sequence between or among different genomes or individuals which has a frequency below 1%.
The term xe2x80x9chaplotypexe2x80x9d refers to a combination of alleles present in an individual or a sample. In the context of the present invention a haplotype preferably refers to a combination of biallelic marker alleles found in a given individual and which may be associated with a phenotype.
The term xe2x80x9cpolymorphismxe2x80x9d as used herein refers to the occurrence of two or more alternative genomic sequences or alleles between or among different genomes or individuals. xe2x80x9cPolymorphicxe2x80x9d refers to the condition in which two or more variants of a specific genomic sequence can be found in a population. A xe2x80x9cpolymorphic sitexe2x80x9d is the locus at which the variation occurs. A single nucleotide polymorphism is a single base pair change. Typically a single nucleotide polymorphism is the replacement of one nucleotide by another nucleotide at the polymorphic site. Deletion of a single nucleotide or insertion of a single nucleotide, also give rise to single nucleotide polymorphisms. In the context of the present invention xe2x80x9csingle nucleotide polymorphismxe2x80x9d preferably refers to a single nucleotide substitution. Typically, between different genomes or between different individuals, the polymorphic site may be occupied by two different nucleotides.
The terms xe2x80x9cbiallelic polymorphismxe2x80x9d and xe2x80x9cbiallelic markerxe2x80x9d are used interchangeably herein to refer to a polymorphism having two alleles at a fairly high frequency in the population, preferably a single nucleotide polymorphism. A xe2x80x9cbiallelic marker allelexe2x80x9d refers to the nucleotide variants present at a biallelic marker site. Typically the frequency of the less common allele of the biallelic markers of the present invention has been validated to be greater than 1%, preferably the frequency is greater than 10%, more preferably the frequency is at least 20% (i.e. heterozygosity rate of at least 0.32), even more preferably the frequency is at least 30% (i.e. heterozygosity rate of at least 0.42). A biallelic marker wherein the frequency of the less common allele is 30% or more is termed a xe2x80x9chigh quality biallelic marker.xe2x80x9d
The location of nucleotides in a polynucleotide with respect to the center of the polynucleotide are described herein in the following manner. When a polynucleotide has an odd number of nucleotides, the nucleotide at an equal distance from the 3xe2x80x2 and 5xe2x80x2 ends of the polynucleotide is considered to be xe2x80x9cat the centerxe2x80x9d of the polynucleotide, and any nucleotide immediately adjacent to the nucleotide at the center, or the nucleotide at the center itself is considered to be xe2x80x9cwithin 1 nucleotide of the center.xe2x80x9d With an odd number of nucleotides in a polynucleotide any of the five nucleotides positions in the middle of the polynucleotide would be considered to be within 2 nucleotides of the center, and so on. When a polynucleotide has an even number of nucleotides, there would be a bond and not a nucleotide at the center of the polynucleotide. Thus, either of the two central nucleotides would be considered to be xe2x80x9cwithin 1 nucleotide of the centerxe2x80x9d and any of the four nucleotides in the middle of the polynucleotide would be considered to be xe2x80x9cwithin 2 nucleotides of the centerxe2x80x9d, and so on. For polymorphisms which involve the substitution, insertion or deletion of 1 or more nucleotides, the polymorphism, allele or biallelic marker is xe2x80x9cat the centerxe2x80x9d of a polynucleotide if the difference between the distance from the substituted, inserted, or deleted polynucleotides of the polymorphism and the 3xe2x80x2 end of the polynucleotide, and the distance from the substituted, inserted, or deleted polynucleotides of the polymorphism and the 5xe2x80x2 end of the polynucleotide is zero or one nucleotide. If this difference is 0 to 3, then the polymorphism is considered to be xe2x80x9cwithin 1 nucleotide of the center.xe2x80x9d If the difference is 0 to 5, the polymorphism is considered to be xe2x80x9cwithin 2 nucleotides of the center.xe2x80x9d If the difference is 0 to 7, the polymorphism is considered to be xe2x80x9cwithin 3 nucleotides of the center,xe2x80x9d and so on. For polymorphisms which involve the substitution, insertion or deletion of 1 or more nucleotides, the polymorphism, allele or biallelic marker is xe2x80x9cat the centerxe2x80x9d of a polynucleotide if the difference between the distance from the substituted, inserted, or deleted polynucleotides of the polymorphism and the 3xe2x80x2 end of the polynucleotide, and the distance from the substituted, inserted, or deleted polynucleotides of the polymorphism and the 5xe2x80x2 end of the polynucleotide is zero or one nucleotide. If this difference is 0 to 3, then the polymorphism is considered to be xe2x80x9cwithin 1 nucleotide of the center.xe2x80x9d If the difference is 0 to 5, the polymorphism is considered to be xe2x80x9cwithin 2 nucleotides of the center.xe2x80x9d If the difference is 0 to 7, the polymorphism is considered to be xe2x80x9cwithin 3 nucleotides of the center,xe2x80x9d and so on.
The term xe2x80x9cupstreamxe2x80x9d is used herein to refer to a location which, is toward the 5xe2x80x2 end of the polynucleotide from a specific reference point.
The terms xe2x80x9cbase pairedxe2x80x9d and xe2x80x9cWatson and Crick base pairedxe2x80x9d are used interchangeably herein to refer to nucleotides which can be hydrogen bonded to one another be virtue of their sequence identities in a manner like that found in double-helical DNA with thymine or uracil residues linked to adenine residues by two hydrogen bonds and cytosine and guanine residues linked by three hydrogen bonds (See Stryer, L., Biochemistry, 4th edition, 1995).
The terms xe2x80x9ccomplementaryxe2x80x9d or xe2x80x9ccomplement thereofxe2x80x9d are used herein to refer to the sequences of polynucleotides which is capable of forming Watson and Crick base pairing with another specified polynucleotide throughout the entirety of the complementary region. This term is applied to pairs of polynucleotides based solely upon their sequences and not any particular set of conditions under which the two polynucleotides would actually bind.
A xe2x80x9cpromoterxe2x80x9d refers to a DNA sequence recognized by the synthetic machinery of the cell required to initiate the specific transcription of a gene.
A sequence which is xe2x80x9coperably linkedxe2x80x9d to a regulatory sequence such as a promoter means that said regulatory element is in the correct location and orientation in relation to the nucleic acid to control RNA polymerase initiation and expression of the nucleic acid of interest.
As used herein, the term xe2x80x9coperably linkedxe2x80x9d refers to a linkage of polynucleotide elements in a functional relationship. For instance, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence. More precisely, two DNA molecules (such as a polynucleotide containing a promoter region and a polynucleotide encoding a desired polypeptide or polynucleotide) are said to be xe2x80x9coperably linkedxe2x80x9d if the nature of the linkage between the two polynucleotides does not (1) result in the introduction of a frame-shift mutation or (2) interfere with the ability of the polynucleotide containing the promoter to direct the transcription of the coding polynucleotide.
The terms xe2x80x9cdisease involving the metabolic conversion of xenobioticsxe2x80x9d refers to susceptibility to a condition or to a condition linked to any of the genes listed in Table 11 (A-B). xe2x80x9cDisease involving the metabolic conversion of xenobioticsxe2x80x9d further refers to a condition involving the biotransformation of drugs and other xenobiotics such as environmental chemicals, food toxins, plant metabolites, carcinogens and industrial chemicals. Such conditions include susceptibility to the toxic or carcinogenic effect of exogenous compounds. xe2x80x9cDisease involving the metabolic conversion of xenobioticsxe2x80x9d also refers to disorders in the metabolism of some endogenous compounds such as the metabolism of steroids, vitamins, fatty acids and eicosanoids such as leukotrienes involving any of the drug-metabolizing enzymes shown in Table 11(A-B). xe2x80x9cDisease involving the metabolic conversion of xenobioticsxe2x80x9d includes, but is not limited to, disorders involving the cytochrome P450 enzyme family, the flavin containing monooxygenases, glucoronidation, the metabolism of glutathione, the pentose pathway and the generation of NADPH.
The term xe2x80x9cdisease involving arachidonic acid metabolismxe2x80x9d refers to a condition linked to disturbances in expression, production or cellular response to eicosanoids such as prostaglandins, thromboxanes, prostacyclins, leukotrienes or hydroperoxyeicosaetrenoic acids. A disease involving arachidonic acid metabolism further refers to a condition involving one or several enzymes of the distinct enzyme systems contributing to arachidonate metabolism including particularly the 5-lipoxygenase pathway. xe2x80x9cDiseases involving arachidonic acid metabolismxe2x80x9d also include chronic inflammatory diseases, acute allergic inflammation and inflammatory conditions such as pain, fever, hypersensitivity, asthma, psoriasis and arthritis. xe2x80x9cDiseases involving arachidonic acid metabolismxe2x80x9d also include disorders in platelet function, blood pressure, thrombosis, renal function, host defense mechanism, hemostasis, smooth muscle tone, male infertility, primary dysmenorrhea, disorders in parturition, and disorders in tissue injury repair, as well as disorders in cellular function and development. xe2x80x9cDiseases involving arachidonic acid metabolismxe2x80x9d also include diseases such as gastrointestinal ulceration, coronary and cerebrovascular syndromes, glomerular immune injury and cancer. Preferably the terms xe2x80x9cdisease involving arachidonic acid metabolismxe2x80x9d refer to a disease including diseases such as cancer, prostate cancer, breast cancer, psoriasis and inflammatory diseases. Preferably the terms xe2x80x9cdisease involving arachidonic acid metabolismxe2x80x9d refer to a disease involving the 5-lipoxygenase pathway and the biosynthesis of the leukotrienes. More preferably the terms xe2x80x9cdisease involving arachidonic acid metabolismxe2x80x9d refer to a disease involving the synthesis of leukotriene C4 (LTC4) and refers to disturbances in expression, activity or function of the human MGST-II enzyme.
As used herein the term xe2x80x9cDME-related biallelic markerxe2x80x9d relates to a set of biallelic markers located in or in the vicinity of the genes disclosed in Table 11(A-B) and further relates to biallelic markers in linkage disequilibrium with the biallelic markers disclosed in Table 11(A-B). The term DME-related biallelic marker encompasses all of the biallelic markers disclosed in Table 11(A-B).
The invention also concerns MGST-II-related biallelic markers. The term xe2x80x9cMGST-II-related biallelic markerxe2x80x9d is used interchangeably herein to relate to all biallelic markers in linkage disequilibrium with the biallelic markers of the MGST-II gene. The term MGST-II-related biallelic marker includes both the genic and non-genic biallelic markers described in Table 3.
The term xe2x80x9cnon-genicxe2x80x9d is used herein to describe MGST-II-related biallelic markers, as well as polynucleotides and primers which occur outside the nucleotide positions shown in the human MGST-II genomic sequence of SEQ ID No. 485. The term xe2x80x9cgenicxe2x80x9d is used herein to describe MGST-II-related biallelic markers as well as polynucleotides and primers which do occur in the nucleotide positions shown in the human MGST-II genomic sequence of SEQ ID No. 485.
The terms xe2x80x9cagent acting on arachidonic acid metabolismxe2x80x9d refers to a drug or a compound modulating the activity or concentration of one or several enzymes of the distinct enzyme systems contributing to arachidonate metabolism including particularly the 5-lipoxygenase pathway. xe2x80x9cAgent acting on arachidonic acid metabolismxe2x80x9d also refers to compounds modulating the formation and action of the eicosanoids including particularly the leukotrienes.
The terms xe2x80x9cresponse to a drugxe2x80x9d refer to drug efficacy, including but not limited to ability to metabolize a therapeutic compound, to the ability to convert a pro-drug to an active drug, and to the pharmacokinetics (absorption, distribution, elimination) and the pharmacodynamics (receptor-related) of a drug in an individual.
The terms xe2x80x9cresponse to an agent acting on arachidonic acid metabolismxe2x80x9d refer to drug efficacy, including but not limited to ability to metabolize a compound, to the ability to convert a pro-drug to an active drug, and to the pharmacokinetics (absorption, distribution, elimination) and the pharmacodynamics (receptor-related) of a drug in an individual.
The terms xe2x80x9cside effects to a drugxe2x80x9d refer to adverse effects of therapy resulting from extensions of the principal pharmacological action of the drug or to idiosyncratic adverse reactions resulting from an interaction of the drug with unique host factors. xe2x80x9cSide effects to a drugxe2x80x9d include, but are not limited to, adverse reactions such as dermatologic, hematologic or hepatologic toxicities and further includes gastric and intestinal ulceration, disturbance in platelet function, renal injury, generalized urticaria, bronchoconstriction, hypotension, and shock.
The terms xe2x80x9cside effects to an agent acting on arachidonic acid metabolismxe2x80x9d refer to adverse effects of therapy resulting from extensions of the principal pharmacological action of the drug or to idiosyncratic adverse reactions resulting from an interaction of the drug with unique host factors. The terms xe2x80x9cside effects to an agent acting on arachidonic acid metabolismxe2x80x9d include, but are not limited to, adverse reactions such as dermatologic, hematologic or hepatologic toxicities.
The term xe2x80x9csequence described in Table 11(A-B)xe2x80x9d is used herein to refer to the entire collection of nucleotide sequences or any individual sequence defined in Table 11(A-B). The SEQ ID that contains each xe2x80x9csequence described in Table 11(A-B)xe2x80x9d is provided in the column labeled, xe2x80x9cSEQ ID NO.xe2x80x9d The column labeled xe2x80x9cGenexe2x80x9d indicates the gene for which the marker is in closest physical proximity, an indication of whether the markers have been validated by microsequencing (with a Y indicating that the markers have been validated by microsequencing and an N indicating that it has not), and an indication of the identity and frequency of the least common allele determined by genotyping (with a blank left to indicate that the frequency has not yet been reported for some markers). The frequencies were determined from DNA samples collected from a random US Caucasian population.
The term xe2x80x9csequence described in Table 11Bxe2x80x9d is used herein to refer to the entire collection of nucleotide sequences or any individual sequence defined in Table 11B. The SEQ ID that contains each xe2x80x9csequence described in Table 11Bxe2x80x9d is provided in the column labeled, xe2x80x9cSEQ ID NO.xe2x80x9d The column labeled xe2x80x9cGenexe2x80x9d indicates the gene for which the marker is in closest physical proximity, an indication of whether the markers have been validated by microsequencing (with a Y indicating that the markers have been validated by microsequencing and an N indicating that it has not), and an indication of the identity and frequency of the least common allele determined by genotyping (with a blank left to indicate that the frequency has not yet been reported for some markers). The frequencies were determined from DNA samples collected from a random US Caucasian population. The xe2x80x9cBiallelic Marker location in SEQ ID No.xe2x80x9d indicates the biallelic marker location within the 47 nucleotide sequence. In Table 11B, this location is 24 for all of the markers.
The term xe2x80x9csequence described in Table 12xe2x80x9d is used herein to refer to the entire collection of nucleotide sequences or any individual sequence defined in Table 12. The SEQ ID that contains each xe2x80x9csequence described in Table 12xe2x80x9d is provided in the column labeled, xe2x80x9cSEQ ID NO.xe2x80x9d The range of nucleotide positions within the Sequence ID of which each sequence consists is provided in the same row as the Sequence ID in a column labeled, xe2x80x9cPOSITION RANGE OF PREFERRED SEQUENCExe2x80x9d. It should be noted that some of the Sequence ID numbers have multiple sequence ranges listed, because they contain multiple xe2x80x9csequences described in Table 12.xe2x80x9d Unless otherwise noted the term xe2x80x9csequence described in Table 12xe2x80x9d is to be construed as encompassing sequences that contain either of the two alleles listed in the columns labeled, xe2x80x9c1ST ALLELExe2x80x9d and xe2x80x9c2ND ALLELExe2x80x9d at the position identified in field  less than 222 greater than  of the allele feature in the appended Sequence Listing for each Sequence ID number referenced in Table 12.
The term xe2x80x9csequence described in Table 13xe2x80x9d is used herein to refer to the entire collection of nucleotide sequences or any individual sequence defined in Table 13. Unless otherwise noted, the xe2x80x9csequences described in Table 13xe2x80x9d consist of the entire sequence of each Sequence ID provided in the column labeled, xe2x80x9cSEQ ID NO.xe2x80x9d Also unless otherwise noted the term xe2x80x9csequence described in Table 13xe2x80x9d is to be construed as encompassing sequences that contain either of the two alleles listed in the columns labeled, xe2x80x9cORIGINAL ALLELExe2x80x9d and xe2x80x9cALTERNATIVE ALLELExe2x80x9d at the position identified in field  less than 222 greater than  of the allele feature in the appended Sequence Listing for each Sequence ID number referenced in Table 13.
The term xe2x80x9csequence described in Table 14xe2x80x9d is used herein to refer to the entire collection of nucleotide sequences or any individual sequence defined in Table 14. Unless otherwise noted, the xe2x80x9csequences described in Table 14xe2x80x9d consist of the entire sequence of each Sequence ID provided in the column labeled, xe2x80x9cSEQ ID NO.xe2x80x9d Also unless otherwise noted the term xe2x80x9csequence described in Table 14xe2x80x9d is to be construed as encompassing sequences that contain either of the two alleles listed in the columns labeled, xe2x80x9c1ST ALLELExe2x80x9d and xe2x80x9c2ND ALLELExe2x80x9d at the position identified in field  less than 222 greater than  of the allele feature in the appended Sequence Listing for each Sequence ID number referenced in Table 14.
The term xe2x80x9csequence described in Table 15xe2x80x9d is used herein to refer to the entire collection of nucleotide sequences or any individual sequence defined in Table 15. The SEQ ID that contains each xe2x80x9csequence described in Table 15xe2x80x9d is provided in the column labeled, xe2x80x9cSEQ ID NO.xe2x80x9d The range of nucleotide positions within the Sequence ID of which each sequence consists is provided in the same row as the Sequence ID in a column labeled, xe2x80x9cPOSITION RANGE OF PREFERRED SEQUENCExe2x80x9d. It should be noted that some of the Sequence ID numbers have multiple sequence ranges listed, because they contain multiple xe2x80x9csequences described in Table 15.xe2x80x9d
The term xe2x80x9csequence described in Table 16xe2x80x9d is used herein to refer to the entire collection of nucleotide sequences or any individual sequence defined in Table 16. The SEQ ID that contains each xe2x80x9csequence described in Table 16xe2x80x9d is provided in the column labeled, xe2x80x9cSEQ IDxe2x80x9d The range of nucleotide positions within the Sequence ID of which half of the sequences consists is provided in the same row as the Sequence ID in a column labeled, xe2x80x9cPOSITION RANGE OF MICROSEQUENCING PRIMERSxe2x80x9d. The remaining half of the sequences described in Table 16 are complementary to the range of nucleotide positions within the Sequence ID provided in the same row as the Sequence ID in a column labeled, xe2x80x9cCOMPLEMENTARY POSITION RANGE OF MICROSEQUENCING PRIMERSxe2x80x9d.
The term xe2x80x9csequence described in Table 17xe2x80x9d is used herein to refer to the entire collection of nucleotide sequences or any individual sequence defined in Table 17. The SEQ ID that contains each xe2x80x9csequence described in Table 17xe2x80x9d is provided in the column labeled, xe2x80x9cSEQ IDxe2x80x9d The range of nucleotide positions within the Sequence ID of which half of the sequences consists is provided in the same row as the Sequence ID in a column labeled, xe2x80x9cPOSITION RANGE OF AMPLIFICATION PRIMERSxe2x80x9d. The remaining half of the sequences described in Table 17 are complementary to the range of nucleotide positions within the Sequence ID provided in the same row as the Sequence ID in a column labeled, xe2x80x9cCOMPLEMENTARY POSITION RANGE OF AMPLIFICATION PRIMERSxe2x80x9d.
The term xe2x80x9csequence described in Table 18xe2x80x9d is used herein to refer to the entire collection of nucleotide sequences or any individual sequence defined in Table 18. The SEQ ID that contains each xe2x80x9csequence described in Table 18xe2x80x9d is provided in the column labeled, xe2x80x9cSEQ IDxe2x80x9d. The range of nucleotide positions within the Sequence ID of which each sequence consists is provided in the same row as the Sequence ID in a column labeled, xe2x80x9cPOSITION RANGE OF PROBESxe2x80x9d. The sequences which are complementary to the ranges listed in the column labeled, xe2x80x9cPOSITION RANGE OF PROBESxe2x80x9d are also encompassed by the term, xe2x80x9csequence described in Table 18.xe2x80x9d Unless otherwise noted the term xe2x80x9csequence described in Table 18xe2x80x9d is to be construed as encompassing sequences that contain either of the two alleles listed in the allele feature in the sequence listing.
The terms xe2x80x9cbiallelic marker described in Tablexe2x80x9d and xe2x80x9callele described in Tablexe2x80x9d are used herein to refer to any or all alleles which are listed in the allele feature in the appended Sequence Listing for each Sequence ID number referenced in the particular Table being mentioned.
Variants and Fragments
The invention also relates to variants and fragments of the polynucleotides described herein, particularly of a MGST-II gene containing one or more biallelic markers according to the invention.
Variants of polynucleotides, as the term is used herein, are polynucleotides that differ from a reference polynucleotide. A variant of a polynucleotide may be a naturally occurring variant such as a naturally occurring allelic variant, or it may be a variant that is not known to occur naturally. Such non-naturally occurring variants of the polynucleotide may be made by mutagenesis techniques, including those applied to polynucleotides, cells or organisms. Generally, differences are limited so that the nucleotide sequences of the reference and the variant are closely similar overall and, in many regions, identical. Variants of polynucleotides according to the invention include, without being limited to, nucleotide sequences which are at least 95% identical, preferably at least 99% identical, more particularly at least 99.5% identical, and most preferably at least 99.8% identical to a polynucleotide selected from the group consisting of the polynucleotides of a sequence from any sequence in the Sequence Listing as well as sequences which are complementary thereto or to any polynucleotide fragment of at least 8 consecutive nucleotides of a sequence from any sequence in the Sequence Listing. Nucleotide changes present in a variant polynucleotide may be silent, which means that they do not alter the amino acids encoded by the polynucleotide. However, nucleotide changes may also result in amino acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the reference sequence. The substitutions, deletions or additions may involve one or more nucleotides. The variants may be altered in coding or non-coding regions or both. Alterations in the coding regions may produce conservative or non-conservative amino acid substitutions, deletions or additions. In the context of the present invention, particularly preferred embodiments are those in which the polynucleotides encode polypeptides which retain substantially the same biological function or activity as the mature MGST-II protein, or those in which the polynucleotides encode polypeptides which maintain or increase a particular biological activity, while reducing a second biological activity. A polynucleotide fragment is a polynucleotide having a sequence that is entirely the same as part but not all of a given nucleotide sequence, preferably the nucleotide sequence of a MGST-II gene, and variants thereof. The fragment can be a portion of an exon or of an intron of a MGST-II gene. It can also be a portion of the regulatory regions of MGST-II, preferably of the promoter sequence of the MGST-II gene. Such fragments may be xe2x80x9cfree-standingxe2x80x9d, i.e. not part of or fused to other polynucleotides, or they may be comprised within a single larger polynucleotide of which they form a part or region. Indeed, several of these fragments may be present within a single larger polynucleotide.
Identity Between Nucleic Acids or Polypeptides
The terms xe2x80x9cpercentage of sequence identityxe2x80x9d and xe2x80x9cpercentage homologyxe2x80x9d are used interchangeably herein to refer to comparisons among polynucleotides and polypeptides, and are determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. Homology is evaluated using any of the variety of sequence comparison algorithms and programs known in the art. Such algorithms and programs include, but are by no means limited to, TBLASTN, BLASTP, FASTA, TFASTA, and CLUSTALW (Pearson and Lipman, Proc. Natl. Acad. Sci. 85(8):2444-2448, 1988; Altschul et al., J. Mol. Biol. 215(3):403-410,1990; Thompson et al., Nucleic Acids Res. 22(2):4673-4680, 1994; Higgins et al., Methods Enzymol. 266:383-402, 1996; Altschul et al., Nature Genetics 3:266-272, 1993, the disclosures of which are incorporated herein by reference in their entireties). In a particularly preferred embodiment, protein and nucleic acid sequence homologies are evaluated using the Basic Local Alignment Search Tool (xe2x80x9cBLASTxe2x80x9d) which is well known in the art (See, e.g., Karlin and Altschul,. Proc. Natl. Acad. Sci. USA 87:2267-2268, 1990; Altschul et al., J. Mol. Biol. 215(3):403-410, 1990; Altschul et al., Nature Genetics 3:266-272, 1993; Altschul et al., Nuc. Acids Res. 25:3389-3402, 1997, the disclosures of which are incorporated herein by reference in their entireties). In particular, five specific BLAST programs are used to perform the following task:
(1) BLASTP and BLAST3 compare an amino acid query sequence against a protein sequence database;
(2) BLASTN compares a nucleotide query sequence against a nucleotide sequence database;
(3) BLASTX compares the six-frame conceptual translation products of a query nucleotide sequence (both strands) against a protein sequence database;
(4) TBLASTN compares a query protein sequence against a nucleotide sequence database translated in all six reading frames (both strands); and
(5) TBLASTX compares the six-frame translations of a nucleotide query sequence against the six-frame translations of a nucleotide sequence database.
The BLAST programs identify homologous sequences by identifying similar segments, which are referred to herein as xe2x80x9chigh-scoring segment pairs,xe2x80x9d between a query amino or nucleic acid sequence and a test sequence which is preferably obtained from a protein or nucleic acid sequence database. High-scoring segment pairs are preferably identified (i.e., aligned) by means of a scoring matrix, many of which are known in the art. Preferably, the scoring matrix used is the BLOSUM62 matrix (Gonnet et al., Science 256:1443-1445, 1992; Henikoff and Henikoff, Proteins 17:49-61, 1993, the disclosures of which are incorporated herein by reference in their entireties). Less preferably, the PAM or PAM250 matrices may also be used (See, e.g., Schwartz and Dayhoff, eds., Matrices for Detecting Distance Relationships: Atlas of Protein Sequence and Structure, Washington:National Biomedical Research Foundation, 1978, the disclosure of which is incorporated herein by reference in its entirety). The BLAST programs evaluate the statistical significance of all high-scoring segment pairs identified, and preferably selects those segments which satisfy a user-specified threshold of significance, such as a user-specified percent homology. Preferably, the statistical significance of a high-scoring segment pair is evaluated using the statistical significance formula of Karlin (see, e.g., Karlin and Altschul, 1990).
Stringent Hybridization Conditions
By way of example and not limitation, procedures using conditions of high stringency are as follows: Prehybridization of filters containing DNA is carried out for 8 h to overnight at 65xc2x0 C. in buffer composed of 6xc3x97SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA, and 500 xcexcg/ml denatured salmon sperm DNA. Filters are hybridized for 48 h at 65xc2x0 C., the preferred hybridization temperature, in prehybridization mixture containing 100 xcexcg/ml denatured salmon sperm DNA and 5-20xc3x97106 cpm of 32P-labeled probe. Alternatively, the hybridization step can be performed at 65xc2x0 C. in the presence of SSC buffer, 1xc3x97SSC corresponding to 0.15M NaCl and 0.05 M Na citrate. Subsequently, filter washes can be done at 37xc2x0 C. for 1 h in a solution containing 2xc3x97SSC, 0.01% PVP, 0.01% Ficoll, and 0.01% BSA, followed by a wash in 0.1xc3x97SSC at 50xc2x0 C. for 45 min. Alternatively, filter washes can be performed in a solution containing 2xc3x97SSC and 0.1% SDS, or 0.5xc3x97SSC and 0.1% SDS, or 0.1xc3x97SSC and 0.1% SDS at 68xc2x0 C. for 15 minute intervals. Following the wash steps, the hybridized probes are detectable by autoradiography. Other conditions of high stringency which may be used are well known in the art and as cited in Sambrook et al., 1989; and Ausubel et al., 1989. These hybridization conditions are suitable for a nucleic acid molecule of about 20 nucleotides in length. There is no need to say that the hybridization conditions described above are to be adapted according to the length of the desired nucleic acid, following techniques well known to the one skilled in the art. The suitable hybridization conditions may for example be adapted according to the teachings disclosed in the book of Hames and Higgins (NucleicAcid Hybridization: A Practical Approach, IRL Press, Oxford, 1985) or in Sambrook et al. (Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989), the disclosures of which are incorporated herein by reference in their entireties.
I. Biallelic Markers and Polynucleotides Comprising Biallelic Markers
A. Polynucleotides of the Present Invention
The present invention encompasses polynucleotides for use as primers and probes in the methods of the invention. These polynucleotides may consist of, consist essentially of, or comprise a contiguous span of nucleotides of a sequence from any sequence in the Sequence Listing as well as sequences which are complementary thereto (xe2x80x9ccomplements thereofxe2x80x9d). The xe2x80x9ccontiguous spanxe2x80x9d maybe at least 8, 10, 12, 15, 18, 20, 25, 35, 40, 50, 70, 80, 100, 250, 500 or 1000 nucleotides in length, to the extent that a contiguous span of these lengths is consistent with the lengths of the particular Sequence ID. It should be noted that the polynucleotides of the present invention are not limited to having the exact flanking sequences surrounding the polymorphic bases which, are enumerated in the Sequence Listing. Rather, it will be appreciated that the flanking sequences surrounding the biallelic markers, or any of the primers of probes of the invention which, are more distant from the markers, may be lengthened or shortened to any extent compatible with their intended use and the present invention specifically contemplates such sequences. It will be appreciated that the polynucleotides referred to in the Sequence Listing may be of any length compatible with their intended use. Also the flanking regions outside of the contiguous span need not be homologous to native flanking sequences which actually occur in human subjects. The addition of any nucleotide sequence, which is compatible with the nucleotides intended use is specifically contemplated. The contiguous span may optionally include the DME-related biallelic marker in said sequence. Biallelic markers generally consist of a polymorphism at one single base position. Each biallelic marker therefore corresponds to two forms of a polynucleotide sequence which, when compared with one another, present a nucleotide modification at one position. Usually, the nucleotide modification involves the substitution of one nucleotide for another. Optionally either the original or the alternative allele of the biallelic markers disclosed in Table 13, or the first or second allele disclosed in Table 12 and 13 may be specified as being present at the DME-related biallelic marker. Optionally, the biallelic markers may be specified as 12-421-135, 12-442-133, 12-449-63, 12-454-242, 12-463-230, 12-462-199, 10-430-287, 12-718-432, 12-269-301, 2-13-398, 2-28-132, 2-39-27, 245-155, 24-391, 12-345-410, 10-358-353, 10-360-190, 10-365-374, 10-367-58, 12-468-424, 12-481-293, 12-499-86, 12-500-217, 12-511-101, 12-586-443, 12-593-287, 12-795-383, 10-494-332, 12-659-251, 12-912-419, 12-914-28, 12-624-307 which consist of more complex polymorphisms including insertions/deletions of at least one nucleotide. Optionally either the original or the alternative allele of these biallelic markers may be specified as being present at the DME-related biallelic marker. Preferred polynucleotides may consist of, consist essentially of, or comprise a contiguous span of nucleotides of a sequence from SEQ ID No. 929-961 well as sequences which are complementary thereto. The xe2x80x9ccontiguous spanxe2x80x9d may be at least 8, 10, 12, 15, 18, 20, 25, 35, 40, 50, 70, 80, 100, 250, 500 or 1000 nucleotides in length, to the extent that a contiguous span of these lengths is consistent with the lengths of the particular Sequence ID. The contiguous span may optionally comprise a biallelic marker selected from the group consisting of biallelic markers 12-421-135, 12-442-133, 12-449-63, 12-454-242, 12-463-230, 12-462-199, 10-430-287, 12-718-432, 12-269-301, 2-13-398, 2-28-132, 2-39-27, 2-45-155, 2-4-391, 12-345-410, 10-358-353, 10-360-190, 10-365-374, 10-367-58, 12-468-424, 12-281-293, 12-499-86, 12-500-217, 12-511-101, 12-586-443, 12-593-287, 12-795-383, 10-494-332, 12-659-251, 12-912-419, 12-914-28, 12-624-307.
The preferred polynucleotides of the invention include the sequence ranges included in any one the sequence ranges of Tables 12, and 15 to 18 individually or in groups consisting of all the possible combinations of the ranges of included in Tables 12, and 15 to 18. The preferred polynucleotides of the invention also include fragments of at least 8, 10, 12, 15, 18, 20, 25, 35, 40, 50, 70, 80, 100, 250, 500 or 1000 consecutive nucleotides of the sequence ranges included in any one of the sequence ranges of Tables 12, and 15 to 18 to the extent that fragments of these lengths are consistent with the lengths of the particular sequence range. The preferred polynucleotides of the invention also include fragments of at least 8, 10, 12, 15, 18, 20, 25, 35, 40, 50, 70, 80, 100, 250, 500 or 1000 consecutive nucleotides of the sequence complementary to the sequence ranges included in any one of the sequence ranges of Tables 12, and 15 to 18 to the extent that fragments of these lengths are consistent with the lengths of the particular sequence range.
Preferred polynucleotides of the invention include isolated, purified or recombinant polynucleotides comprising a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of a sequence selected from the group consisting of the sequences of SEQ ID Nos. 485-487, 490-493, 495, 496, 498-523, 930-934, and 965, and the complements thereof.
Particularly preferred polynucleotides of the invention include isolated, purified or recombinant polynucleotides comprising a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No. 485, wherein said contiguous span comprises at least 1, 2, 3, 4, 5 or 10 of the following nucleotide positions of SEQ ID No. 485: 1 to 7667, 7726 to 20264, 20365 to 36918, 36991 to 45180, 45263 to 45741, and 45980 to 49327, and the complements thereof. Other particularly preferred polynucleotides of the present invention include isolated, purified or recombinant polynucleotides comprising a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No. 486, wherein said contiguous span comprises at least 1, 2, 3, 4, 5 or 10 of nucleotide positions 1 to 198 of SEQ ID No. 486 and the complements thereof. Other particularly preferred polynucleotides of the present invention include isolated, purified or recombinant polynucleotides comprising a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No. 487, wherein said contiguous span comprises at least 1, 2, 3, 4, 5 or 10 of nucleotide positions 1 to 198 of SEQ ID No. 487 and the complements thereof. Other particularly preferred polynucleotides of the present invention include isolated, purified or recombinant polynucleotides comprising a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No. 490, wherein said contiguous span comprises at least 1, 2, 3, 4, 5 or 10 of nucleotide positions 1 to 198 of SEQ ID No. 490 and the complements thereof. Other preferred polynucleotides of the present invention include polynucleotides comprising, consisting of, or consisting essentially of a nucleotide sequence of SEQ ID No. 491.
Particularly preferred polynucleotides of the present invention include purified, isolated or recombinant polynucleotides comprising a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of a sequence selected from the group consisting of SEQ ID SEQ ID Nos. 3, 5, 9, 13-15, 25, 31, 33, 37, 38, 41, 323, 345, 351-353, 357, 377, and 380, or the complements thereof; or more preferably SEQ ID Nos. 496, 498, 502, 506-508, 518, 524, 526, 530, 531, 534, 816, 838, 844-846, 850, 870, and 873, or the complements thereof, wherein said span includes a MGST-II-related biallelic marker. Optionally either allele of the biallelic markers described above in the definition of MGST-II-related biallelic marker is specified as being present at the MGST-II-related biallelic marker.
Additional preferred polynucleotides of the invention include isolated, purified or recombinant polynucleotide comprising a contiguous span of at least 12 nucleotides of SEQ ID No. 485 or the complementary sequence thereof, wherein said contiguous span comprises a nucleotide selected from the group consisting of a T at position 36971, a C at position 45214 or a T at position 45741 of SEQ ID No. 485. Additional preferred polynucleotides of the invention include isolated, purified or recombinant polynucleotides comprising a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides from a sequence of SEQ ID No. 486, wherein said contiguous span comprises a T at position 426, a C at position 478 or a T at position 526 of SEQ ID No. 486; or the complement thereof. Additional preferred polynucleotides of the invention include isolated, purified or recombinant polynucleotides comprising a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides from a sequence of SEQ ID No. 487, wherein said contiguous span comprises a T at position 325, a C at position 378 or a T at position 426 of SEQ ID No. 487; or the complements thereof.
Table 3(A-D) contains a list of preferred MGST-II-related biallelic markers. Each marker is described by indicating its Marker ID, the position of the marker in the SEQ ID and the two most common alleles.
The invention also relates to polynucleotides that hybridize, under conditions of high or intermediate stringency, to a polynucleotide of a sequence from any sequence in the Sequence Listing as well as sequences, which are complementary thereto. Preferably such polynucleotides are at least 20, 25, 35, 40, 50, 70, 80, 100, 250, 500 or 1000 nucleotides in length, to the extent that a polynucleotide of these lengths is consistent with the lengths of the particular Sequence ID. Preferred polynucleotides comprise a DME-related biallelic marker. Optionally either the original or the alternative allele of the biallelic markers disclosed in Table 13 may be specified as being present at the DME-related biallelic marker. Conditions of high and intermediate stringency are further described in xe2x80x9cMethods of Genotyping DNA Samples for Biallelic Markers-Hybridization Assay Methods.xe2x80x9d
The present invention further embodies isolated, purified, and recombinant polynucleotides which encode MGST-II polypeptides comprising a contiguous span of at least 6 amino acids, preferably at least 8 to 10 amino acids, more preferably at least 12, 15, 20, 25, 30, 40, 50, or 100 amino acids of SEQ ID No. 488. The present invention further embodies isolated, purified, and recombinant polynucleotides which encode the variant MGST-II polypeptides of SEQ ID Nos. 488 and 489. The present invention further embodies isolated, purified, and recombinant polynucleotides which encode a variant MGST-II polypeptide comprising a contiguous span of at least 6 amino acids, preferably at least 8 to 10 amino acids, more preferably at least 12, 15, 20, 25, 30, 40, 50, or 100 amino acids of SEQ ID No. 489. The present invention further embodies isolated, purified, and recombinant polynucleotides which encode polypeptides comprising a contiguous span of at least 6 amino acids, preferably at least 8 to 10 amino acids, more preferably at least 12, 15, 20, 25, 30, 40, 50, or 100 amino acids of SEQ ID No. 488 wherein said contiguous span comprises a His residue at amino acid position 93. The present invention further embodies isolated, purified, and recombinant polynucleotides which encode polypeptides comprising a contiguous span of at least 6 amino acids, preferably at least 8 to 10 amino acids, more preferably at least 12, 15, 20, 25, 30, 40, 50, or 100 amino acids of amino acid positions 1-108 of SEQ ID No. 488.
The primers of the present invention may be designed from the disclosed sequences for any method known in the art. A preferred set of primers is fashioned such that the 3xe2x80x2 end of the contiguous span of identity with the sequences of the Sequence Listing is present at the 3xe2x80x2 end of the primer. Such a configuration allows the 3xe2x80x2 end of the primer to hybridize to a selected nucleic acid sequence and dramatically increases the efficiency of the primer for amplification or sequencing reactions. In a preferred set of primers the contiguous span is found in one of the sequences described in Table 15. Allele specific primers may be designed such that a biallelic marker is at the 3xe2x80x2 end of the contiguous span and the contiguous span is present at the 3xe2x80x2 end of the primer. Such allele specific primers tend to selectively prime an amplification or sequencing reaction so long as they are used with a nucleic acid sample that contains one of the two alleles present at a biallelic marker. The 3xe2x80x2 end of primer of the invention may be located within or at least 2, 4, 6, 8, 10, 12, 15, 18, 20, 25, 50, 100, 250, 500, or 1000, to the extent that this distance is consistent with the particular Sequence ID, nucleotides upstream of a DME-related biallelic marker in said sequence or at any other location which is appropriate for their intended use in sequencing, amplification or the location of novel sequences or markers. A list of preferred amplification primers is disclosed in Table 17. Primers with their 3xe2x80x2 ends located 1 nucleotide upstream of a DME-related biallelic marker have a special utility as microsequencing assays. Preferred microsequencing primers are described in Table 16.
The probes of the present invention may be designed from the disclosed sequences for any method known in the art, particularly methods which allow for testing if a particular sequence or marker disclosed herein is present. A preferred set of probes may be designed for use in the hybridization assays of the invention in any manner known in the art such that they selectively bind to one allele of a biallelic marker, but not the other under any particular set of assay conditions. Preferred hybridization probes may consists of, consist essentially of, or comprise a contiguous span which ranges in length from 8, 10, 12, 15, 18 or 20 to 25, 35, 40, 50, 60, 70, or 80 nucleotides, or be specified as being 12, 15, 18, 20, 25, 35, 40, or 50 nucleotides in length and including a DME-related biallelic marker of said sequence. Optionally the original allele or alternative allele disclosed in Table 13 and 14 may be specified as being present at the biallelic marker site. Optionally, said biallelic marker may be within 6, 5, 4, 3, 2, or 1 nucleotides of the center of the hybridization probe or at the center of said probe. A particularly preferred set of hybridization probes is disclosed in Table 18 or a sequence complementary thereto.
Any of the polynucleotides of the present invention can be labeled, if desired, by incorporating a label detectable by spectroscopic, photochemical, biochemical, immunochemical, or chemical means. For example, useful labels include radioactive substances, fluorescent dyes or biotin. Preferably, polynucleotides are labeled at their 3xe2x80x2 and 5xe2x80x2 ends. A label can also be used to capture the primer, so as to facilitate the immobilization of either the primer or a primer extension product, such as amplified DNA, on a solid support. A capture label is attached to the primers or probes and can be a specific binding member which forms a binding pair with the solid""s phase reagent""s specific binding member (e.g. biotin and streptavidin). Therefore depending upon the type of label carried by a polynucleotide or a probe, it may be employed to capture or to detect the target DNA. Further, it will be understood that the polynucleotides, primers or probes provided herein, may, themselves, serve as the capture label. For example, in the case where a solid phase reagent""s binding member is a nucleic acid sequence, it may be selected such that it binds a complementary portion of a primer or probe to thereby immobilize the primer or probe to the solid phase. In cases where a polynucleotide probe itself serves as the binding member, those skilled in the art will recognize that the probe will contain a sequence or xe2x80x9ctailxe2x80x9d that is not complementary to the target. In the case where a polynucleotide primer itself serves as the capture label, at least a portion of the primer will be free to hybridize with a nucleic acid on a solid phase. DNA Labeling techniques are well known to the skilled technician.
Any of the polynucleotides, primers and probes of the present invention can be conveniently immobilized on a solid support. Solid supports are known to those skilled in the art and include the walls of wells of a reaction tray, test tubes, polystyrene beads, magnetic beads, nitrocellulose strips, membranes, microparticles such as latex particles, sheep (or other animal) red blood cells, duracytes(copyright) and others. The solid support is not critical and can be selected by one skilled in the art. Thus, latex particles, microparticles, magnetic or non-magnetic beads, membranes, plastic tubes, walls of microtiter wells, glass or silicon chips, sheep (or other suitable animal""s) red blood cells and duracytes are all suitable examples. Suitable methods for immobilizing nucleic acids on solid phases include ionic, hydrophobic, covalent interactions and the like. A solid support, as used herein, refers to any material which is insoluble, or can be made insoluble by a subsequent reaction. The solid support can be chosen for its intrinsic ability to attract and immobilize the capture reagent. Alternatively, the solid phase can retain an additional receptor which has the ability to attract and immobilize the capture reagent. The additional receptor can include a charged substance that is oppositely charged with respect to the capture reagent itself or to a charged substance conjugated to the capture reagent. As yet another alternative, the receptor molecule can be any specific binding member which is immobilized upon (attached to) the solid support and which has the ability to immobilize the capture reagent through a specific binding reaction. The receptor molecule enables the indirect binding of the capture reagent to a solid support material before the performance of the assay or during the performance of the assay. The solid phase thus can be a plastic, derivatized plastic, magnetic or non-magnetic metal, glass or silicon surface of a test tube, microtiter well, sheet, bead, microparticle, chip, sheep (or other suitable animal""s) red blood cells, duracytes(copyright) and other configurations known to those of ordinary skill in the art. The polynucleotides of the invention can be attached to or immobilized on a solid support individually or in groups of at least 2, 5, 8, 10, 12, 15, 20, or 25 distinct polynucleotides of the inventions to a single solid support. In addition, polynucleotides other than those of the invention may attached to the same solid support as one or more polynucleotides of the invention.
Any polynucleotide provided herein may be attached in overlapping areas or at random locations on the solid support. Alternatively the polynucleotides of the invention may be attached in an ordered array wherein each polynucleotide is attached to a distinct region of the solid support which does not overlap with the attachment site of any other polynucleotide. Preferably, such an ordered array of polynucleotides is designed to be xe2x80x9caddressablexe2x80x9d where the distinct locations are recorded and can be accessed as part of an assay procedure. Addressable polynucleotide arrays typically comprise a plurality of different oligonucleotide probes that are coupled to a surface of a substrate in different known locations. The knowledge of the precise location of each polynucleotides location makes these xe2x80x9caddressablexe2x80x9d arrays particularly useful in hybridization assays. Any addressable array technology known in the aft can be employed with the polynucleotides of the invention. One particular embodiment of these polynucleotide arrays is known as the Genechips(trademark), and has been generally described in U.S. Pat. No. 5,143,854; PCT publications WO 90/15070 and 92/10092, the disclosures of which are incorporated herein by reference in their entirety. These arrays may generally be produced using mechanical synthesis methods or light directed synthesis methods, which incorporate a combination of photolithographic methods and solid phase oligonucleotide synthesis (Fodor et al., Science, 251:767-777, 1991). The immobilization of arrays of oligonucleotides on solid supports has been rendered possible by the development of a technology generally identified as xe2x80x9cVery Large Scale Immobilized Polymer Synthesisxe2x80x9d (VLSIPS(trademark)) in which, typically, probes are immobilized in a high density array on a solid surface of a chip. Examples of VLSIPS(trademark) technologies are provided in U.S. Pat. Nos. 5,143,854 and 5,412,087 and in PCT Publications WO 90/15070, WO 92/10092 and WO 95/11995, the disclosures of which are incorporated herein by reference in their entirety, which describe methods for forming oligonucleotide arrays through techniques such as light-directed synthesis techniques. In designing strategies aimed at providing arrays of nucleotides immobilized on solid supports, further presentation strategies were developed to order and display the oligonucleotide arrays on the chips in an attempt to maximize hybridization patterns and sequence information. Examples of such presentation strategies are disclosed in PCT Publications WO 94/12305, WO 94/11530, WO 97/29212 and WO 97/31256, the disclosures of which are incorporated herein by reference in their entirety.
Oligonucleotide arrays may comprise at least one of the sequences selected from the group consisting of SEQ ID Nos. 1-38, 40-54, 56-353, 355-463, and 465-487, and the sequences complementary thereto; or more preferably SEQ ID Nos. 485-487, 494-531, 533-547, 549-846, 848-956, and 958-977, and the sequences complementary thereto; or a fragment thereof of at least 8, 10, 12, 15, 18, 20, 25, 35, 40, 50, 70, 80, 100, 250, 500 or 1000 consecutive nucleotides, to the extent that fragments of these lengths is consistent with the lengths of the particular Sequence ID, for determining whether a sample contains one or more alleles of the biallelic markers of the present invention. Oligonucleotide arrays may also comprise at least one of the sequences selected from the group consisting of SEQ ID Nos. 1-38, 40-54, 56-353, 355-463, and 465-487, and the sequences complementary thereto; or more preferably SEQ ID Nos. 485-487, 494-531, 533-547, 549-846, 848-956, and 958-977, and the sequences complementary thereto; or a fragment thereof of at least 8, 10, 12, 15, 18, 20, 25, 35, 40, 50, 70, 80, 100, 250, 500 or 1000 consecutive nucleotides, to the extent that fragments of these lengths is consistent with the lengths of the particular Sequence ID, for amplifying one or more alleles of the biallelic markers of Table 11. In other embodiments, arrays may also comprise at least one of the sequences selected from the group consisting of SEQ ID Nos. 1-38, 40-54, 56-353, 355-463, and 465-487, and the sequences complementary thereto; or more preferably SEQ ID Nos. 485-487, 494-531, 533-547, 549-846, 848-956, and 958-977, and the sequences complementary thereto; or a fragment thereof of at least 8, 10, 12, 15, 18, 20, 25, 35, 40, 50, 70, 80, 100, 250, 500 or 1000 consecutive nucleotides, to the extent that fragments of these lengths is consistent with the lengths of the particular Sequence ID, for conducting microsequencing analyses to determine whether a sample contains one or more alleles of the biallelic markers of the invention. In still further embodiments, the oligonucleotide array may comprise at least one of the sequences selecting from the group consisting of SEQ ID Nos. 1-38, 40-54, 56-353, 355-463, and 465-487, and the sequences complementary thereto; or more preferably SEQ ID Nos. 485-487, 494-531, 533-547, 549-846, 848-956, and 958-977, and the sequences complementary thereto; or a fragment thereof of at least 8, 10, 12, 15, 18, 20, 25, 35, 40, 50, 70, 80, 100, 250, 500 or 1000 nucleotides in length, to the extent that fragments of these lengths is consistent with the lengths of the particular Sequence ID, for determining whether a sample contains one or more alleles of the biallelic markers of the present invention.
The present invention also encompasses diagnostic kits comprising one or more polynucleotides of the invention, optionally with a portion or all of the necessary reagents and instructions for genotyping a test subject by determining the identity of a nucleotide at a DME-related biallelic marker. The polynucleotides of a kit may optionally be attached to a solid support, or be part of an array or addressable array of polynucleotides. The kit may provide for the determination of the identity of the nucleotide at a marker position by any method known in the art including, but not limited to, a sequencing assay method, a microsequencing assay method, a hybridization assay method, or an allele specific amplification method. Optionally such a kit may include instructions for scoring the results of the determination with respect to the test subjects"" risk of contracting a diseases involving the metabolic conversion of xenobiotics, or likely response to a drug, or chances of suffering from side effects to a drug, including hepatotoxicity.
B. Genomic Sequences of the MGST-II Gene and Biallelic Markers
The present invention encompasses the genomic sequence of the MGST-II gene of SEQ ID No. 485. The MGST-II genomic sequences comprise exons and introns. Particularly preferred genomic sequences of the MGST-II gene of the invention include isolated, purified or recombinant polynucleotides comprising a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No. 485, wherein said contiguous span comprises at least 1, 2, 3, 4, 5 or 10 of the following nucleotide positions of SEQ ID No. 485: 1 to 7667, 7726 to 20264, 20365 to 36918, 36991 to 45180, 45263 to 45741, and 45980 to 49327, and the complements thereof. The nucleic acids defining the MGST-II intronic polynucleotides may be used as oligonucleotide primers or probes in order to detect the presence of a copy of the MGST-II gene in a test sample, or alternatively in order to amplify a target nucleotide sequence within the MGST-II sequences.
The present invention further provides MGST-II intron and exon polynucleotide sequences including biallelic markers. Particularly preferred polynucleotides of the present invention include purified, isolated or recombinant polynucleotides comprising a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of a sequence of SEQ ID No. 485; or the complements thereof; wherein said span includes a MGST-II-related biallelic marker. Preferred polynucleotides comprise at least one biallelic marker selected from the group consisting of biallelic markers 10-286-289, 10-287-116, 10-286-375, 12-425-57, 12-421-135, 12-421-140, 10-523-232, 10-289-201, 10-290-37, 10-290-326 and 10-290-328. The present invention also provides polynucleotides which, may be used as primers and probes in order to amplify fragments carrying biallelic markers or in order to detect biallelic marker alleles.
Regulatory Sequences
The genomic sequence of the MGST-II gene contains regulatory sequences both in the non-coding 5xe2x80x2-flanking region and in the non-coding 3xe2x80x2-flanking region that border the MGST-II transcribed region containing the 5 exons of this gene. 5xe2x80x2-regulatory sequences of the MGST-II gene comprise the polynucleotide sequences located between the nucleotide in position 1 and the nucleotide in position 7466 of the nucleotide sequence of SEQ ID No. 485. 3xe2x80x2-regulatory sequences of the MGST-II gene comprise the polynucleotide sequences located between the nucleotide in position 45980 and the nucleotide in position 49327 of the nucleotide sequence of SEQ II) No. 485. Particularly preferred regulatory polynucleotides of the present invention include isolated, purified or recombinant polynucleotides comprising a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No. 485, wherein said contiguous span comprises at least 1, 2, 3, 4, 5 or 10 of the following nucleotide positions of SEQ ID No. 485: 1 to 7466 and 45966 to 49312; and the complements thereof.
The promoter activity of the regulatory regions contained in the MGST-II genomic sequence of polynucleotide sequence of SEQ ID No. 485 can be assessed by any method known in the art. Methods for identifying the polynucleotide fragments of SEQ ID No. 485 involved in the regulation of the expression of the MGST-II gene are well-known to those skilled in the art (see Sambrook et al., Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989). An example of a typical method, that can be used, involves a recombinant vector carrying a reporter gene and genomic sequences from the MGST-II genomic sequence of SEQ ID No. 485. Briefly, the expression of the reporter gene (for example beta galactosidase or chloramphenicol acetyl transferase) is detected when placed under the control of a biologically active polynucleotide fragment. Genomic sequences located upstream of the first exon of the MGST-II gene may be cloned into any suitable promoter reporter vector, such as the pSEAP-Basic, pSEAP-Enhancer, pxcex2gal-Basic, pxcex2gal-Enhancer, or pEGFP-1 Promoter Reporter vectors available from Clontech, or pGL2-basic or pGL3-basic promoterless luciferase reporter gene vector from Promega. Each of these promoter reporter vectors include multiple cloning sites positioned upstream of a reporter gene encoding a readily assayable protein such as secreted alkaline phosphatase, luciferase, beta galactosidase, or green fluorescent protein. The sequences upstream the first MGST-II exon are inserted into the cloning sites upstream of the reporter gene in both orientations and introduced into an appropriate host cell. The level of reporter protein is assayed and compared to the level obtained with a vector lacking an insert in the cloning site. The presence of an elevated expression level in the vector containing the insert with respect to the control vector indicates the presence of a promoter in the insert.
Promoter sequences within the 5xe2x80x2 non-coding regions of the MGST-II gene may be further defined by constructing nested 5xe2x80x2 and/or 3xe2x80x2 deletions using conventional techniques such as Exonuclease III or appropriate restriction endonuclease digestion. The resulting deletion fragments can be inserted into the promoter reporter vector to determine whether the deletion has reduced or obliterated promoter activity, such as described, for example, by Coles et al. (Hum. Mol. Genet., 7:791-800, 1998). In this way, the boundaries of the promoters may be defined. If desired, potential individual regulatory sites within the promoter may be identified using site directed mutagenesis or linker scanning to obliterate potential transcription factor binding sites within the promoter individually or in combination. The effects of these mutations on transcription levels may be determined by inserting the mutations into cloning sites in promoter reporter vectors. This type of assays are well known to those skilled in the art and are further described in WO 97/17359, U.S. Pat. No. 5,374,544, EP 582 796, U.S. Pat. Nos. 5,698,389, 5,643,746, 5,502,176, and 5,266,488.
The activity and the specificity of the promoter of the MGST-II gene can further be assessed by monitoring the expression level of a detectable polynucleotide operably linked to the MGST-II promoter in different types of cells and tissues. The detectable polynucleotide may be either a polynucleotide that specifically hybridizes with a predefined oligonucleotide probe, or a polynucleotide encoding a detectable protein, including a MGST-II polypeptide or a fragment or a variant thereof. This type of assay is well known to those skilled in the art and is described in U.S. Pat. Nos. 5,502,176 and 5,266,488 for example.
Polynucleotides carrying the regulatory elements located both at the 5xe2x80x2 end and at the 3xe2x80x2 end of the MGST-II coding region may be advantageously used to control the transcriptional and translational activity of an heterologous polynucleotide of interest, said polynucleotide being heterologous as regards to the MGST-II regulatory region.
Thus, the present invention also concerns a purified, isolated, and recombinant nucleic acid comprising a polynucleotide which, is selected from the group consisting of, the polynucleotide sequences located between the nucleotide in position 1 and the nucleotide in position 7466 of the nucleotide sequence of SEQ ID No. 485; or a sequence complementary thereto or a biologically active fragment thereof.
By a xe2x80x9cbiologically activexe2x80x9d fragment of SEQ ID No. 485 according to the present invention is intended a polynucleotide comprising or alternatively consisting of a fragment of said polynucleotide which is functional as a regulatory region for expressing a recombinant polypeptide or a recombinant polynucleotide in a recombinant cell host.
For the purpose of the invention, a nucleic acid or polynucleotide is xe2x80x9cfunctionalxe2x80x9d as a regulatory region for expressing a recombinant polypeptide or a recombinant polynucleotide if said regulatory polynucleotide contains nucleotide sequences which contain transcriptional and translational regulatory information, and such sequences are xe2x80x9coperably linkedxe2x80x9d to nucleotide sequences which encode the desired polypeptide or the desired polynucleotide.
The regulatory polynucleotides according to the invention may be advantageously part of a recombinant expression vector that may be used to express a coding sequence in a desired host cell or host organism.
C. MGST-II cDNA Comprising Biallelic Markers and Variant MGST-II cDNA
The present invention provides a MGST-II cDNA of SEQ ID No. 486. The Open Reading Frame encoding the MGST-II protein spans from the nucleotide in position 202 to the nucleotide in position 642 of the polynucleotide sequence of SEQ ID No. 486. The cDNA of SEQ ID No. 486 also includes a 5xe2x80x2-UTR region and a 3xe2x80x2-UTR region.
Particularly preferred cDNA polynucleotides of the present invention include purified, isolated or recombinant polynucleotides comprising a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of a sequence of SEQ ID No. 486, or the complements thereof, wherein said span includes a MGST-II-related biallelic marker. Preferred cDNA fragments comprise a biallelic marker selected from the group consisting of 10-286-289 (position 98), 10-286-345 (position 153), 10-286-375 (position 183), 10-523-232 (position 426), 10-289-201 (position 478) and 10-290-37 (position 526). Additional preferred polynucleotides of the invention include isolated, purified or recombinant polynucleotides comprising a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides from a sequence of SEQ ID No. 486, wherein said contiguous span comprises a T at position 426, a C at position 478 or a T at position 526 of SEQ ID No. 486; or the complement thereof. Most biallelic polymorphisms represent silent nucleotide substitutions but biallelic marker 10-289-201 is associated with an amino acid change in the corresponding MGST-II polypeptide (TYR replaced by ARG in position 93 of the polypeptide). Moreover, one biallelic marker allele of marker 10-290-37 is associated with a stop codon and the corresponding variant cDNA encodes a truncated MGST-II polypeptide including amino acids 1 to 108.
The present invention further provides a variant MGST-II cDNA of SEQ ID No. 487. corresponding to an alternative splicing form which results in the deletion of exon 2. This alternative splicing of MGST-II yields the variant MGST-II polypeptide of SEQ ID No. 489. MGST-II polypeptides of the present invention are further described below. Preferred cDNAs of the invention include isolated, purified or recombinant polynucleotides comprising a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides from a sequence of SEQ ID No. 487; or the complements thereof. Additional preferred polynucleotides of the invention include isolated, purified or recombinant polynucleotides comprising a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides from a sequence of SEQ ID No. 487, wherein said contiguous span comprises a T at position 325, a C at position 378 or a T at position 426 of SEQ ID No. 487; or the complements thereof. The new exon 1/exon 3 junction sequence of the splice variant MGST-II cDNA, more particularly the nucleotide sequence comprised between the nucleotide in position 106 and the nucleotide in position 374 of the nucleic acid of SEQ ID No. 486 corresponds to the nucleotide sequence of an EST that has been obtained from a human cDNA library. Polynucleotides comprising this EST of a sequence from SEQ ID No. 490 are also part of the invention.
The above disclosed polynucleotides that contain the coding sequence of the MGST-II gene and of MGST-II variants may be expressed in a desired host cell or a desired host organism, when this polynucleotide is placed under the control of suitable expression signals. The expression signals may be either the expression signals contained in the regulatory regions in the MGST-II gene of the invention or in contrast the signals may be exogenous regulatory nucleic sequences. Such a polynucleotide, when placed under the suitable expression signals, may also be inserted in a vector for its expression and/or amplification.
Another preferred cDNA fragment comprises the 5xe2x80x2-UTR region (regulatory) beginning at position 1 and ending at position 201 of SEQ ID Nos. 486 and 487. Preferably said 5xe2x80x2-UTR region comprises a biallelic marker selected from the group consisting of biallelic markers 10-286-289, 10-286-345 and 10-286-375. Particularly preferred 5xe2x80x2-UTR polynucleotides of the present invention include isolated, purified or recombinant polynucleotides comprising a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No. 486, wherein said contiguous span comprises at least 1, 2, 3, 4, 5 or 10 of nucleotide positions 1 to 198 of SEQ ID No. 486; and the complements thereof. The 5xe2x80x2-end sequence of the MGST-II cDNA, more particularly the nucleotide sequence comprised between the nucleotide in position 1 and the nucleotide in position 357 of the nucleic acid of SEQ ID No. 486 corresponds to the nucleotide sequence of a 5xe2x80x2-EST that has been obtained from a human cDNA library. Polynucleotides comprising this 5xe2x80x2-EST of a sequence from SEQ ID No. 490 are also part of the invention.
The polynucleotide disclosed above that contains the coding sequence of the MGST-II gene of the invention may be expressed in a desired host cell or a desired host organism, when this polynucleotide is placed under the control of suitable expression signals. The expression signals may be either the expression signals contained in the regulatory regions in the MGST-II gene of the invention or may be exogenous regulatory nucleic sequences. Such a polynucleotide, when placed under the suitable expression signals, may also be inserted in a vector for its expression.
A further object of the invention consists of an isolated polynucleotide comprising:
a) a nucleic acid comprising a regulatory nucleotide sequence from a sequence of SEQ ID No. 485;
b) a polynucleotide encoding a desired polypeptide or a nucleic acid of interest, operably linked to the nucleic acid defined in (a) above; and
c) Optionally, a nucleic acid comprising a 5xe2x80x2-UTR regulatory polynucleotide, preferably a 5xe2x80x2-UTR regulatory polynucleotide sequence of a sequence of SEQ ID No. 486.
The polypeptide encoded by the nucleic acid described above may be of various nature or origin, encompassing proteins of prokaryotic or eukaryotic origin. Among the polypeptides expressed under the control of a MGST-II regulatory region, there may be cited bacterial, fungal or viral antigens. Also encompassed are eukaryotic proteins such as intracellular proteins, for example xe2x80x9chouse keepingxe2x80x9d proteins, membrane-bound proteins, for example receptors, and secreted proteins, for example cytokines. In a specific embodiment, the desired polypeptide may be the MGST-II protein, especially the proteins of the amino acid sequence of SEQ ID Nos. 488 and 489.
The desired nucleic acids encoded by the above described polynucleotide, usually a RNA molecule, may be complementary to a desired coding polynucleotide, for example to the MGST-II coding sequence, and thus useful as an antisense polynucleotide.
Such a polynucleotide may be included in a recombinant expression vector in order to express the desired polypeptide or the desired nucleic acid in host cell or in a host organism.
D. Polynucleotide Constructs, Recombinant Vectors, Host Cells and Transgenic Animals
Polynucleotide Constructs
The terms xe2x80x9cpolynucleotide constructxe2x80x9d and xe2x80x9crecombinant polynucleotidexe2x80x9d are used interchangeably herein to refer to linear or circular, purified or isolated polynucleotides that have been artificially designed and which comprise at least two nucleotide sequences that are not found as contiguous nucleotide sequences in their initial natural environment.
1. DNA Constructs for Expressing the MGST-II Gene in Recombinant Host Cells and in Transgenic Animals
In order to study the physiological and phenotype consequences of a lack of synthesis of the MGST-II protein, both at the cellular level and at the multicellular organism level, in particular as regards to disorders related to abnormal cell proliferation, notably cancers, the invention also encompasses DNA constructs and recombinant vectors enabling a conditional expression of a specific allele of the MGST-II genomic sequence or cDNA.
A first preferred DNA construct is based on the tetracycline resistance operon tet from E. coli transposon Tn110 for controlling the MGST-II gene expression, such as described by Gossen et al. (Science, 268:1766-1769, 1995). Such a DNA construct contains seven tet operator sequences from Tn10 (tetop) that are fused to either a minimal promoter or a 5xe2x80x2-regulatory sequence of the MGST-II gene, said minimal promoter or said MGST-II regulatory sequence being operably linked to a polynucleotide of interest that codes either for a sense or an antisense oligonucleotide or for a polypeptide, including a MGST-II polypeptide or a peptide fragment thereof. This DNA construct is functional as a conditional expression system for the nucleotide sequence of interest when the same cell also comprises a nucleotide sequence coding for either the wild type (tTA) or the mutant (rTA) repressor fused to the activating domain of viral protein VP16 of herpes simplex virus, placed under the control of a promoter, such as the HCMVIE1 enhancer/promoter or the MMTV-LTR. Indeed, a preferred DNA construct of the invention will comprise both the polynucleotide containing the tet operator sequences and the polynucleotide containing a sequence coding for the tTA or the rTA repressor. In the specific embodiment wherein the conditional expression DNA construct contains the sequence encoding the mutant tetracycline repressor rTA, the expression of the polynucleotide of interest is silent in the absence of tetracycline and induced in its presence.
2. DNA Constructs Allowing Homologous Recombination: Replacement Vectors
A second preferred DNA construct will comprise, from 5xe2x80x2-end to 3xe2x80x2-end: (a) a first nucleotide sequence that is comprised in the MGST-II genomic sequence; (b) a nucleotide sequence comprising a positive selection marker, such as the marker for neomycine resistance (neo); and (c) a second nucleotide sequence that is comprised in the MGST-II genomic sequence, and is located on the genome downstream the first MGST-II nucleotide sequence (a).
In a preferred embodiment, this DNA construct also comprises a negative selection marker located upstream the nucleotide sequence (a) or downstream the nucleotide sequence (c). Preferably, the negative selection marker consists of the thymidine kinase (tk) gene (Thomas et al., Cell, 44:419-428, 1986), the hygromycine beta gene (Te Riele et al., Nature, 348:649-651, 1990), the hprt gene (Van der Lugt et al., Gene, 105:263-267, 1991; Reid et al., Proc. Natl. Acad. Sci. USA, 87:4299-4303, 1990) or the Diphteria toxin A fragment (Dt-A) gene (Nada et al., Cell, 73:1125-1135, 1993; Yagi et al., Proc. Natl; Acad. Sci. USA, 87:9918-9922, 1990). Preferably, the positive selection marker is located within a MGST-II exon sequence so as to interrupt the sequence encoding a MGST-II protein. These replacement vectors are further described by Mansour et al. (Nature, 336:348-352, 1988) and Koller et al. (Ann. Rev. Immunol., 10:705-730, 1992). The first and second nucleotide sequences (a) and (c) may be indifferently located within a MGST-II regulatory sequence, an intronic sequence, an exon sequence or a sequence containing both regulatory and/or intronic and/or exon sequences. The size of the nucleotide sequences (a) and (c) is ranging from 1 to 50 kb, preferably from 1 to 10 kb, more preferably from 2 to 6 kb and most preferably from 2 to 4 kb.
3. DNA Constructs Allowing Homologous Recombination: Cre-loxP System
These new DNA constructs make use of the site specific recombination system of the P1 phage. The P1 phage possesses a recombinase called Cre which, interacts specifically with a 34 base pairs loxP site. The loxP site is composed of two palindromic sequences of 13 bp separated by a 8 bp conserved sequence (Hoess et al., Nucleic Acids Res., 14:2287-2300, 1986). The recombination by the Cre enzyme between two loxP sites having an identical orientation leads to the deletion of the DNA fragment.
The Cre-loxP system used in combination with a homologous recombination technique has been first described by Gu et al. (Cell, 73:1155-1164, 1993). Briefly, a nucleotide sequence of interest to be inserted in a targeted location of the genome harbors at least two loxP sites in the same orientation and located at the respective ends of a nucleotide sequence to be excised from the recombinant genome. The excision event requires the presence of the recombinase (Cre) enzyme within the nucleus of the recombinant cell host. The recombinase enzyme may be brought at the desired time either by (a) incubating the recombinant cell hosts in a culture medium containing this enzyme, by injecting the Cre enzyme directly into the desired cell, such as described by Araki et al. (Proc. Natl; Acad. Sci. USA, 92: 160-164, 1995), or by lipofection of the enzyme into the cells, such as described by Baubonis et al. (Nucleic Acids Res., 21:2025-2029, 1993); (b) transfecting the cell host with a vector comprising the Cre coding sequence operably linked to a promoter functional in the recombinant cell host, which promoter being optionally inducible, said vector being introduced in the recombinant cell host, such as described by Gu et al. (Cell, 73:1155-1164, 1993) and Sauer et al. (Proc. Natl; Acad. Sci. USA, 85:5166-5170, 1988); (c) introducing in the genome of the cell host a polynucleotide comprising the Cre coding sequence operably linked to a promoter functional in the recombinant cell host, which promoter is optionally inducible, and said polynucleotide being inserted in the genome of the cell host either by a random insertion event or an homologous recombination event, such as described by Gu et al. (Science, 265:103-106, 1994).
In the specific embodiment wherein the vector containing the sequence to be inserted in the MGST-II gene by homologous recombination is constructed in such a way that selectable markers are flanked by loxP sites of the same orientation, it is possible, by treatment by the Cre enzyme, to eliminate the selectable markers while leaving the MGST-II sequences of interest that have been inserted by an homologous recombination event. Again, two selectable markers are needed: a positive selection marker to select for the recombination event and a negative selection marker to select for the homologous recombination event. Vectors and methods using the Cre-loxP system are further described by Zou et al. (Curr. Biol., 4:1099-1103, 1994).
Thus, a third preferred DNA construct of the invention comprises, from 5xe2x80x2-end to 3xe2x80x2-end: (a) a first nucleotide sequence that is comprised in the MGST-II genomic sequence; (b) a nucleotide sequence comprising a polynucleotide encoding a positive selection marker, said nucleotide sequence comprising additionally two sequences defining a site recognized by a recombinase, such as a loxP site, the two sites being placed in the same orientation; and (c) a second nucleotide sequence that is comprised in the MGST-II genomic sequence, and is located on the genome downstream of the first MGST-II nucleotide sequence (a).
The sequences defining a site recognized by a recombinase, such as a loxP site, are preferably located within the nucleotide sequence (b) at suitable locations bordering the nucleotide sequence for which the conditional excision is sought. In one specific embodiment, two loxP sites are located at each side of the positive selection marker sequence, in order to allow its excision at a desired time after the occurrence of the homologous recombination event.
In a preferred embodiment of a method using the third DNA construct described above, the excision of the polynucleotide fragment bordered by the two sites recognized by a recombinase, preferably two loxP sites, is performed at a desired time, due to the presence within the genome of the recombinant cell host of a sequence encoding the Cre enzyme operably linked to a promoter sequence, preferably an inducible promoter, more preferably a tissue-specific promoter sequence and most preferably a promoter sequence which is both inducible and tissue-specific, such as described by Gu et al. (Science, 265:103-106, 1994).
The presence of the Cre enzyme within the genome of the recombinant cell host may result of the breeding of two transgenic animals, the first transgenic animal bearing the MGST-II-derived sequence of interest containing the loxP sites as described above and the second transgenic animal bearing the Cre coding sequence operably linked to a suitable promoter sequence, such as described by Gu et al. (Science, 265:103-106, 1994).
Spatio-temporal control of the Cre enzyme expression may also be achieved with an adenovirus based vector that contains the Cre gene thus allowing infection of cells, or in vivo infection of organs, for delivery of the Cre enzyme, such as described by Anton et al. (J. Virol., 69:4600-4606, 1995) and Kanegae et al. (Nucleic Acids Res., 23:3816-3821, 1995).
The DNA constructs described above may be used to introduce a desired nucleotide sequence of the invention, preferably a MGST-II genomic sequence or a MGST-II cDNA sequence, and most preferably an altered copy of a MGST-II genomic or cDNA sequence, within a predetermined location of the targeted genome, leading either to the generation of an altered copy of a targeted gene (knock-out homologous recombination) or to the replacement of a copy of the targeted gene by another copy sufficiently homologous to allow an homologous recombination event to occur (knock-in homologous recombination).
Recombinant Vectors
The term xe2x80x9cvectorxe2x80x9d is used herein to designate either a circular or a linear DNA or RNA molecule, which is either double-stranded or single-stranded, and which comprise at least one polynucleotide of interest that is sought to be transferred in a cell host or in a unicellular or multicellular host organism.
The present invention encompasses a family of recombinant vectors that comprise a regulatory polynucleotide derived from the MGST-II genomic sequence, or a coding polynucleotide from the MGST-II genomic sequence. Consequently, the present invention further deals with a recombinant vector comprising either a regulatory polynucleotide comprised in the nucleic acid of SEQ ID Nos. 485 and 486 or a polynucleotide comprising the MGST-II coding sequence or both.
In a first preferred embodiment, a recombinant vector of the invention is used to amplify the inserted polynucleotide derived from a MGST-II genomic sequence selected from the group consisting of the nucleic acids of SEQ ID No. 485 or a MGST-II cDNA, for example the cDNA of SEQ ID Nos. 486 and 487 in a suitable host cell, this polynucleotide being amplified each time the recombinant vector replicates. Generally, a recombinant vector of the invention may comprise any of the polynucleotides described herein, including regulatory sequences and coding sequences, as well as any MGST-II primer or probe as defined above.
A second preferred embodiment of the recombinant vectors according to the invention consists of expression vectors comprising either a regulatory polynucleotide or a coding nucleic acid of the invention, or both. Within certain embodiments, expression vectors are employed to express the MGST-II polypeptide which can be then purified and, for example be used in ligand screening assays or as an immunogen in order to raise specific antibodies directed against the MGST-II protein. In other embodiments, the expression vectors are used for constructing transgenic animals and also for gene therapy. Expression requires that appropriate signals are provided in the vectors, said signals including various regulatory elements, such as enhancers/promoters from both viral and mammalian sources that drive expression of the genes of interest in host cells. Dominant drug selection markers for establishing permanent, stable cell clones expressing the products are generally included in the expression vectors of the invention, as they are elements that link expression of the drug selection markers to expression of the polypeptide.
More particularly, the present invention relates to expression vectors which include nucleic acids encoding a MGST-II protein, preferably the MGST-II protein of the amino acid sequence of SEQ ID Nos. 488 and 489, under the control of a regulatory sequence selected among the MGST-II regulatory polynucleotides of SEQ ID Nos. 485 and 486, or alternatively under the control of an exogenous regulatory sequence. Consequently, preferred expression vectors of the invention are selected from the group consisting of: (a) the MGST-II regulatory sequence comprised therein drives the expression of a coding polynucleotide operably linked thereto; (b) the MGST-II coding sequence is operably linked to regulation sequences allowing its expression in a suitable cell host and/or host organism. Additionally, the recombinant expression vector described above may also comprise a nucleic acid comprising a 5xe2x80x2-regulatory polynucleotide or a 3xe2x80x2-regulatory polynucleotide, preferably a 5xe2x80x2-regulatory polynucleotide or a 3xe2x80x2-regulatory polynucleotide of the MGST-II gene. The MGST-II 5xe2x80x2-regulatory polynucleotide may also comprise the 5xe2x80x2-UTR sequence contained in the nucleotide sequence of SEQ ID Nos. 486 and 487; or a biologically active fragment or variant thereof. The invention also pertains to a recombinant expression vector useful for the expression of the MGST-II coding sequence, wherein said vector comprises any of the MGST-II cDNAs or cDNA variants described above; or fragments thereof.
Some of the elements which, can be found in the vectors of the present invention are described in further detail in the following sections.
1. General Features of the Expression Rectors of the Invention
A recombinant vector according to the invention comprises, but is not limited to, a YAC (Yeast Artificial Chromosome), a BAC (Bacterial Artificial Chromosome), a phage, a phagemid, a cosmid, a plasmid or even a linear DNA molecule which may consist of a chromosomal, non-chromosomal, semi-synthetic and synthetic DNA. Such a recombinant vector can comprise a transcriptional unit comprising an assembly of:
(1) a genetic element or elements having a regulatory role in gene expression, for example promoters or enhancers. Enhancers are cis-acting elements of DNA, usually from about 10 to 300 bp in length that act on the promoter to increase the transcription.
(2) a structural or coding sequence which is transcribed into mRNA and eventually translated into a polypeptide, said structural or coding sequence being operably linked to the regulatory elements described in (1); and
(3) appropriate transcription initiation and termination sequences. Structural units intended for use in yeast or eukaryotic expression systems preferably include a leader sequence enabling extracellular secretion of translated protein by a host cell. Alternatively, when a recombinant protein is expressed without a leader or transport sequence, it may include a N-terminal residue. This residue may or may not be subsequently cleaved from the expressed recombinant protein to provide a final product.
Generally, recombinant expression vectors will include origins of replication, selectable markers permitting transformation of the host cell, and a promoter derived from a highly expressed gene to direct transcription of a downstream structural sequence. The heterologous structural sequence is assembled in appropriate phase with translation initiation and termination sequences, and preferably a leader sequence capable of directing secretion of the translated protein into the periplasmic space or the extracellular medium. In a specific embodiment wherein the vector is adapted for transfecting and expressing desired sequences in mammalian host cells, preferred vectors will comprise an origin of replication in the desired host, a suitable promoter and enhancer, and also any necessary ribosome binding sites, polyadenylation site, splice donor and acceptor sites, transcriptional termination sequences, and 5xe2x80x2-flanking non-transcribed sequences. DNA sequences derived from the SV40 viral genome, for example SV40 origin, early promoter, enhancer, splice and polyadenylation sites may be used to provide the required non-transcribed genetic elements.
The in vivo expression of a MGST-II polypeptide of SEQ ID Nos. 488 and 489 may be useful in order to correct a genetic defect related to the expression of the native gene in a host organism or to the production of a biologically inactive MGST-II protein.
Consequently, the present invention also deals with recombinant expression vectors mainly designed for the in vivo production of the MGST-II polypeptide of SEQ ID Nos. 488 and 489 or fragments or variants thereof by the introduction of the appropriate genetic material in the organism of the patient to be treated. This genetic material may be introduced in vitro in a cell that has been previously extracted from the organism, the modified cell being subsequently reintroduced in the said organism, directly in vivo into the appropriate tissue.
2. Regulatory Elements
Promoters
The suitable promoter regions used in the expression vectors according to the present invention are chosen taking into account the cell host in which the heterologous gene has to be expressed. The particular promoter employed to control the expression of a nucleic acid sequence of interest is not believed to be important, so long as it is capable of directing the expression of the nucleic acid in the targeted cell. Thus, where a human cell is targeted, it is preferable to position the nucleic acid coding region adjacent to and under the control of a promoter that is capable of being expressed in a human cell, such as, for example, a human or a viral promoter.
A suitable promoter may be heterologous with respect to the nucleic acid for which it controls the expression or alternatively can be endogenous to the native polynucleotide containing the coding sequence to be expressed. Additionally, the promoter is generally heterologous with respect to the recombinant vector sequences within which the construct promoter/coding sequence has been inserted.
Promoter regions can be selected from any desired gene using, for example, CAT (chloramphenicol transferase) vectors and more preferably pKK232-8 and pCM7 vectors.
Preferred bacterial promoters are the LacI, LacZ, the T3 or T7 bacteriophage RNA polymerase promoters, the gpt, lambda PR, PL and trp promoters (EP 0036776), the polyhedrin promoter, or the p10 protein promoter from baculovirus (Kit Novagen) (Smith et al., 1983; O""Reilly et al., 1992), the lambda PR promoter or also the trc promoter.
Eukaryotic promoters include CMV immediate early, HSV thymidine kinase, early and late SV40, LTRs from retrovirus, and mouse metallothionein-L. Selection of a convenient vector and promoter is well within the level of ordinary skill in the art.
The choice of a promoter is well within the ability of a person skilled in the field of genetic egineering. For example, one may refer to the book of Sambrook et al. (Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989).
Other Regulatory Elements
Where a cDNA insert is employed, one will typically desire to include a polyadenylation signal to effect proper polyadenylation of the gene transcript. The nature of the polyadenylation signal is not believed to be crucial to the successful practice of the invention, and any such sequence may be employed such as human growth hormone and SV40 polyadenylation signals. Also contemplated as an element of the expression cassette is a terminator. These elements can serve to enhance message levels and to minimize read through from the cassette into other sequences.
The vector containing the appropriate DNA sequence as described above, more preferably MGST-II gene regulatory polynucleotide, a polynucleotide encoding the MGST-II polypeptides of SEQ ID Nos. 488 and 489 or both of them, can be utilized to transform an appropriate host to allow the expression of the desired polypeptide or polynucleotide.
3. Selectable Markers
Such markers would confer an identifiable change to the cell permitting easy identification of cells containing the expression construct. The selectable marker genes for selection of transformed host cells are preferably dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, TRP 1 for S. cerevisiae or tetracycline, rifampicin or ampicillin resistance in E. coli, or levan saccharase for mycobacteria, this latter marker being a negative selection marker.
4. Preferred Vectors
Bacterial Vectors
As a representative but non-limiting example, useful expression vectors for bacterial use can comprise a selectable marker and a bacterial origin of replication derived from commercially available plasmids comprising genetic elements of pBR322 (ATCC 37017). Such commercial vectors include, for example, pKK223-3 (Pharmacia, Uppsala, Sweden), and GEM1 (Promega Biotec, Madison, Wis., USA). Large numbers of other suitable vectors are known to those of skill in the art, and commercially available, such as the following bacterial vectors: pQE70, pQE60, pQE-9 (Qiagen), pbs, pD10, phagescript, psiX174, pbluescript SK, pbsks, pNH8A, pNH16A, pNH18A, pNH46A (Stratagene); ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia); pWLNEO, pSV2CAT, pOG44, pXT1, pSG (Stratagene); pSVK3, pBPV, pMSG, pSVL (Pharmacia); pQE-30 (QLAexpress).
Bacteriophage Vectors
The P1 bacteriophage vector may contain large inserts ranging from about 80 to about 100 kb. The construction of P1 bacteriophage vectors such as p158 or p158/neo8 have been described by Sternberg (Mamm. Genome, 5:397404, 1994). Recombinant P1 clones comprising MGST-II nucleotide sequences may be designed for inserting large polynucleotides of more than 40 kb (Linton et al., J. Clin. Invest., 92:3029-3037, 1993). To generate P1 DNA for transgenic experiments, a preferred protocol is the protocol described by McCormick et al. (Genet. Anal. Tech. Appl., 11:158-164, 1994). Briefly, E. coli (preferably strain NS3529) harboring the P1 plasmid are grown overnight in a suitable broth medium containing 25 xcexcg/ml of kanamycin. The P1 DNA is prepared from the E. coli by alkaline lysis using the Qiagen Plasmid Maxi kit (Qiagen, Chatsworth, Calif., USA), according to the manufacturer""s instructions. The P1 DNA is purified from the bacterial lysate on two Qiagen-tip 500 columns, using the washing and elution buffers contained in the kit. A phenol/chloroform extraction is then performed before precipitating the DNA with 70% ethanol. After solubilizing the DNA in TE (10 mM Tris-HCl, pH 7.4, 1 mM EDTA), the concentration of the DNA is assessed by spectrophotometry.
When the goal is to express a P1 clone comprising MGST-II nucleotide sequences in a transgenic animal, typically in transgenic mice, it is desirable to remove vector sequences from the P1 DNA fragment, for example by cleaving the P1 DNA at rare-cutting sites within the P1 polylinker (SfiI, NotI or SalI). The P1 insert is then purified from vector sequences on a pulsed-field agarose gel, using methods similar using methods similar to those originally reported for the isolation of DNA from YACs (Schedl et al., 1993a; Peterson et al., 1993). At this stage, the resulting purified insert DNA can be concentrated, if necessary, on a Millipore Ultrafree-MC Filter Unit (Millipore, Bedford, Mass., USAxe2x80x9430,000 molecular weight limit) and then dialyzed against microinjection buffer (10 mM Tris-HCl, pH 7.4; 250 xcexcM EDTA) containing 100 mM NaCl, 30 xcexcM spermine, 70 xcexcM spermidine on a microdyalisis membrane (type VS, 0.025 xcexcM from Millipore). The intactness of the purified P1 DNA insert is assessed by electrophoresis on 1% agarose (Sea Kem GTG; FMC Bio-products) pulse-field gel and staining with ethidium bromide.
Baculovirus Vectors
A suitable vector for the expression of the MGST-II polypeptides of SEQ ID Nos. 488 and 489 is a baculovirus vector that can be propagated in insect cells and in insect cell lines. A specific suitable host vector system is the pVL1392/1393 baculovirus transfer vector (Pharmingen) that is used to transfect the SF9 cell line (ATCC Nxc2x0 CRL 1711) which is derived from Spodoptera frugiperda. 
Other suitable vectors for the expression of the MGST-II polypeptides of SEQ ID Nos. 488 and 489 in a baculovirus expression system include those described by Chai et al. (Biotech. Appl. Biochem., 18:259-273, 1993), Vlasak et al. (Eur. J. Biochem., 135: 123-126, 1983) and Lenhard et al. (Gene, 169: 187-190, 1996).
Viral Vectors
Retrovirus vectors and adeno-associated virus vectors are generally understood to be the recombinant gene delivery systems of choice for the transfer of exogenous polynucleotides in vivo, particularly to mammals, including humans. These vectors provide efficient delivery of genes into cells, and the transferred nucleic acids are stably integrated into the chromosomal DNA of the host.
Particularly preferred retroviruses for the preparation or construction of retroviral in vitro or in vitro gene delivery vehicles of the present invention include retroviruses selected from the group consisting of Mink-Cell Focus Inducing Virus, Murine Sarcoma Virus, Reticuloendotheliosis virus and Rous Sarcoma virus. Particularly preferred Murine Leukemia Viruses include the 4070A and the 1504A viruses, Abelson (ATCC No VR-999), Friend (ATCC No VR-245), Gross (ATCC No. VR-590), Rauscher (ATCC No VR-998) and Moloney Murine Leukemia Virus (ATCC No VR-190; PCT Application No WO 94/24298). Particularly preferred Rous Sarcoma Viruses include Bryan high titer (ATCC Nos. VR-334, VR-657, VR-726, VR-659 and VR-728). Other preferred retroviral vectors are those described in Roth et al. (Nature Medicine, 2:985-991, 1996), PCT Application No. WO 93/25234 and PCT Application No. WO 94/06920.
Yet another viral vector system that is contemplated by the invention consists in the adeno-associated virus (AAV). The adeno-associated virus is a naturally occurring defective virus that requires another virus, such as an adenovirus or a herpes virus, as a helper virus for efficient replication and a productive life cycle (Muzyczka et al., Current Topics in Microbiol. Immunol., 158:97-129, 1992). It is also one of the few viruses that may integrate its DNA into non-dividing cells, and exhibits a high frequency of stable integration (McLaughlin et al., Am. J. Hum. Genet., 59: 561-569, 1989). One advantageous feature of AAV derives from its reduced efficacy for transducing primary cells relative to transformed cells. BAC vectors:
The bacterial artificial chromosome (BAC) cloning system (Shizuya et al., 1992) has been developed to stably maintain large fragments of genomic DNA (100-300 kb) in E. coli. A preferred BAC vector consists of pBeloBAC11 vector that has been described by Kim et al. (Genomics, 34:213-218,1996). BAC libraries are prepared with this vector using size-selected genomic DNA that has been partially digested using enzymes that permit ligation into either the Bam HI or HindIII sites in the vector. Flanking these cloning sites are T7 and SP6 RNA polymerase transcription initiation sites that can be used to generate end probes by either RNA transcription or PCR methods. After the construction of a BAC library in E. coli, BAC DNA is purified from the host cell as a supercoiled circle. Converting these circular molecules into a linear form precedes both size determination and introduction of the BACs into recipient cells. The cloning site is flanked by two Not I sites, permitting cloned segments to be excised from the vector by Not I digestion. Alternatively, the DNA insert contained in the pBeloBAC11 vector may be linearized by treatment of the BAC vector with the commercially available enzyme lambda terminase that leads to the cleavage at the unique cosN site, but this cleavage method results in a full length BAC clone containing both the insert DNA and the BAC sequences.
5. Delivery of the Recombinant Vectors
In order to effect expression of the polynucleotides and polynucleotide constructs of the invention, these constructs must be delivered into a cell. This delivery may be accomplished in vitro, as in laboratory procedures for transforming cell lines, or in vivo or ex vivo, as in the treatment of certain diseases states. One mechanism is viral infection where the expression construct is encapsidated in an infectious viral particle. Several non-viral methods for the transfer of polynucleotides into cultured mammalian cells are also contemplated by the present invention, and include, without being limited to, calcium phosphate precipitation (Chen et al., Proc. Natl. Acad. Sci. USA, 94:10756-10761, 1987), DEAE-dextran (Gopal, Mol. Cell. Biol., 5:1188-1190, 1985), electroporation (Tur-Kaspa et al., Mol. Cell. Biol., 6:716-7181986), direct microinjection (Harland et al., 1985), DNA-loaded liposomes (Nicolau et al., 1982; Fraley et al., 1979), and receptor-mediate transfection (Wu and Wu, 1987; 1988). Some of these techniques may be successfully adapted for in vivo or ex vivo use.
Once the expression polynucleotide has been delivered into the cell, it may be stably integrated into the genome of the recipient cell. This integration may be in the cognate location and orientation via homologous recombination (gene replacement) or it may be integrated in a random, non-specific location (gene augmentation). In yet further embodiments, the nucleic acid may be stably maintained in the cell as a separate, episomal segment of DNA. Such nucleic acid segments or xe2x80x9cepisomesxe2x80x9d encode sequences sufficient to permit maintenance and replication independent of or in synchronization with the host cell cycle.
One specific embodiment for a method for delivering a protein or peptide to the interior of a cell of a vertebrate in vivo comprises the step of introducing a preparation comprising a physiologically acceptable carrier and a naked polynucleotide operatively coding for the polypeptide of interest into the interstitial space of a tissue comprising the cell, whereby the naked polynucleotide is taken up into the interior of the cell and has a physiological effect. This is particularly applicable for transfer in vitro but it may be applied to in vivo as well.
Compositions for use in vitro and in vivo comprising a xe2x80x9cnakedxe2x80x9d polynucleotide are described in PCT application No. WO 90/11092 (Vical Inc.) and also in PCT application No. WO 95/11307.
In still another embodiment of the invention, the transfer of a naked polynucleotide of the invention, including a polynucleotide construct of the invention, into cells may be proceeded with a particle bombardment (biolistic), said particles being DNA-coated microprojectiles accelerated to a high velocity allowing them to pierce cell membranes and enter cells without killing them, such as described by Klein et al. (Nature 327:70-73, 1987).
In a further embodiment, the polynucleotide of the invention may be entrapped in a liposome (Ghosh and Bacchawat, Targeting of liposomes to hepatocytes, In: Liver Diseases, Targeted diagnosis and therapy using specific rceptors and ligands, Marcel Dekeker, New York, 87-104, 1991; Wong et al., Gene 10:87-94, 1980; Nicolau et al., Biochim. Biophys. Acta. 721:185-190, 1982).
In a specific embodiment, the invention provides a composition for the in vivo production of the MGST-II protein or polypeptide described herein. It comprises a naked polynucleotide operatively coding for this polypeptide, in solution in a physiologically acceptable carrier, and suitable for introduction into a tissue to cause cells of the tissue to express the said protein or polypeptide.
The amount of vector to be injected to the desired host organism varies according to the site of injection. As an indicative dose, it will be injected between 0.1 and 100 xcexcg of the vector in an animal body, preferably a mammal body, for example a mouse body.
In another embodiment of the invention, the vector may be introduced in vitro in a host cell, preferably in a host cell previously harvested from the animal to be treated and more preferably a somatic cell such as a muscle cell. In a subsequent step, the cell that has been transformed with the vector coding for the desired MGST-II polypeptide or the desired fragment thereof is reintroduced into the animal body in order to deliver the recombinant protein within the body either locally or systemically.
Host Cells
Another embodiment of the invention consists of a host cell that has been transformed or transfected with one of the polynucleotides described therein, and more precisely a polynucleotide either comprising a MGST-II regulatory polynucleotide or the coding sequence of the MGST-II polypeptide having the amino acid sequence of SEQ ID Nos. 488 and 489. The embodiment includes host cells that are transformed (prokaryotic cells) or that are transfected (eukaryotic cells) with a recombinant vector such as one of those described above. Generally, a recombinant host cell of the invention comprises any one of the polynucleotides or the recombinant vectors described therein.
A preferred recombinant host cell according to the invention comprises a polynucleotide selected from the following group of polynucleotides:
a) a purified or isolated nucleic acid encoding a MGST-II polypeptide, or a polypeptide fragment or variant thereof.
b) a purified or isolated nucleic comprising at least 8, preferably at least 15, more preferably at least 25, consecutive nucleotides of the nucleotide sequence SEQ ID No. 485, a nucleotide sequence complementary thereto, or a variant thereof.
c) a purified or isolated nucleic acid comprising at least 8 consecutive nucleotides, preferably at least 15, more preferably at least 25 of the nucleotide sequence SEQ ID Nos. 486 and 487, a nucleotide sequence complementary thereto or a variant thereof.
d) a purified or isolated nucleic acid comprising an exon of the MGST-II gene, a sequence complementary thereto or a fragment or a variant thereof.
e) a purified or isolated nucleic acid comprising a combination of at least two exons of the MGST-II gene, or the sequences complementary thereto wherein the polynucleotides are arranged within the nucleic acid, from the 5xe2x80x2 end to the 3xe2x80x2 end of said nucleic acid, in the same order than in SEQ ID No. 485.
f) a purified or isolated nucleic acid comprising the nucleotide sequence SEQ ID No. 485 or the sequences complementary thereto or a biologically active fragment thereof.
g) a purified or isolated nucleic acid comprising the nucleotide sequence SEQ ID No. 486, or the sequence complementary thereto or a biologically active fragment thereof.
h) a polynucleotide consisting of:
(1) a nucleic acid comprising a regulatory polynucleotide of SEQ ID No. 485 or the sequences complementary thereto or a biologically active fragment thereof
(2) a polynucleotide encoding a desired polypeptide or nucleic acid.
(3) Optionally, a nucleic acid comprising a regulatory polynucleotide of SEQ ID No. 485, or the sequence complementary thereto or a biologically active fragment thereof.
i) a DNA construct as described previously in the present specification.
Another preferred recombinant cell host according to the present invention is characterized in that its genome or genetic background (including chromosome, plasmids) is modified by the nucleic acid coding for the MGST-II polypeptide of SEQ ID Nos. 488 and 489 or fragments or variants thereof.
Preferred host cells used as recipients for the expression vectors of the invention are the following:
a) Prokaryotic host cells: Escherichia coli strains (I.E. DH5-xcex1 strain), Bacillus subtilis, Salmonella typhimurium, and strains from species like Pseudomonas, Streptomyces and Staphylococcus..
b) Eukaryotic host cells: HeLa cells (ATCC Nxc2x0 CCL2; Nxc2x0 CCL2.1; Nxc2x0 CCL2.2), Cv 1 cells (ATCC Nxc2x0 CCL70), COS cells (ATCC Nxc2x0 CRL1650; Nxc2x0 CRL1651), Sf-9 cells (ATCC Nxc2x0 Nxc2x0 CRL1711), C127 cells (ATCC Nxc2x0 CRL-1804), 3T3 (ATCC Nxc2x0 CRL-6361), CHO (ATCC Nxc2x0 CCL-61), human kidney 293.(ATCC Nxc2x0 45504; Nxc2x0 CRL-1573) and BHK (ECACC Nxc2x0 84100501; Nxc2x0 84111301)
c) Other mammalian host cells:
The MGST-II gene expression in mammalian, and typically human, cells may be rendered defective, or alternatively it may be proceeded with the insertion of a MGST-II genomic or cDNA sequence with the replacement of the MGST-II gene counterpart in the genome of an animal cell by a MGST-II polynucleotide according to the invention. These genetic alterations may be generated by homologous recombination events using specific DNA constructs that have been previously described.
One kind of cell host that is commonly used is mammal zygotes, such as murine zygotes. For example, murine zygotes may undergo microinjection with a purified DNA molecule of interest, for example a purified DNA molecule that has previously been adjusted to a concentration range from 1 ng/ml (for BAC inserts) 3 ng/xcexcl (for P1 bacteriophage inserts) in 10 mM Tris-HCl, pH 7.4, 250 xcexcM EDTA containing 100 mM NaCl, 30 xcexcM spermine,and 70 xcexcM spermidine. When the DNA to be microinjected has a large size, polyamines and high salt concentrations can be used in order to avoid mechanical breakage of this DNA, as described by Schedl et al (Nucleic Acids Res. 21:47834787, 1993).
Anyone of the polynucleotides of the invention, including the DNA constructs described herein, may be introduced in an embryonic stem (ES) cell line, preferably a mouse ES cell line. ES cell lines are derived from pluripotent, uncommitted cells of the inner cell mass of pre-implantation blastocysts. Preferred ES cell lines are the following: ES-E14TG2a (ATCC nxc2x0 CRL-1821), ES-D3 (ATCC nxc2x0 CRL1934 and nxc2x0 CRL-11632), YS001 (ATCC nxc2x0 CRL-11776), 36.5 (ATCC nxc2x0 CRL-11116). To maintain ES cells in an uncommitted state, they are cultured in the presence of growth inhibited feeder cells which, provide the appropriate signals to preserve this embryonic phenotype and serve as a matrix for ES cell adherence. Preferred feeder cells consist of primary embryonic fibroblasts that are established from tissue of day 13-day 14 embryos of virtually any mouse strain, that are maintained in culture, such as described by Abbondanzo et al. (Methods in Enzymology, Academic Press, NewYork, 803-823, 1993) and are inhibited in growth by irradiation, such as described by Robertson (xe2x80x9cEmbryo-Derived StemCell Lines,xe2x80x9d E. J. Robertson Ed. Teratocarcinomas and Embrionic Stem Cells: A Practical Approach. IRL Press, Oxford, 71, 1987), or by the presence of an inhibitory concentration of LIF, such as described by Pease and Williams (Exp. Cell. Res. 190:09-211, 1990).
The constructs in the host cells can be used in a conventional manner to produce the gene product encoded by the recombinant sequence.
Following transformation of a suitable host and growth of the host to an appropriate cell density, the selected promoter is induced by appropriate means, such as temperature shift or chemical induction, and cells are cultivated for an additional period.
Cells are typically harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract retained for further purification.
Microbial cells employed in the expression of proteins can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents. Such methods are well known by one skilled in the art.
Transgenic Animals
The terms xe2x80x9ctransgenic animalsxe2x80x9d or xe2x80x9chost animalsxe2x80x9d are used herein designate animals that have their genome genetically and artificially manipulated so as to include one of the nucleic acids according to the invention. Preferred animals are non-human mammals and include those belonging to a genus selected from Mus (e.g. mice), Rattus (e.g. rats) and Oryctogalus (e.g. rabbits) which have their genome artificially and genetically altered by the insertion of a nucleic acid according to the invention.
The transgenic animals of the invention all include within a plurality of their cells a cloned recombinant or synthetic DNA sequence, more specifically one of the purified or isolated nucleic acids comprising a MGST-II coding sequence, a MGST-II regulatory polynucleotide or a DNA sequence encoding an antisense polynucleotide such as described in the present specification.
Preferred transgenic animals according to the invention contains in their somatic cells and/or in their germ line cells a polynucleotide selected from the following group of polynucleotides:
a) a purified or isolated nucleic acid encoding a MGST-II polypeptide, or a polypeptide fragment or variant thereof.
b) a purified or isolated nucleic comprising at least 8, preferably at least 15, more preferably at least 25, consecutive nucleotides of the nucleotide sequence SEQ ID No. 485, a nucleotide sequence complementary thereto.
c) a purified or isolated nucleic acid comprising at least 8 consecutive nucleotides, preferably at least 15, more preferably at least 25 of the nucleotide sequence SEQ ID Nos. 486 and 487, a nucleotide sequence complementary thereto.
d) a purified or isolated nucleic acid comprising an exon of the MGST-II gene, a sequence complementary thereto or a fragment or a variant thereof.
e) a purified or isolated nucleic acid comprising a combination of at least two exons of the MGST-II gene, or the sequences complementary thereto wherein the polynucleotides are arranged within the nucleic acid, from the 5xe2x80x2 end to the 3xe2x80x2 end of said nucleic acid, in the same order than in SEQ ID No. 485.
f) a purified or isolated nucleic acid comprising the nucleotide sequence SEQ ID No. 485 or the sequences complementary thereto or a biologically active fragment thereof.
g) a purified or isolated nucleic acid comprising the nucleotide sequence SEQ ID No. 486, or the sequence complementary thereto or a biologically active fragment thereof.
h) a polynucleotide consisting of:
(1) a nucleic acid comprising a regulatory polynucleotide of SEQ ID No. 485 or the sequences complementary thereto or a biologically active fragment thereof
(2) a polynucleotide encoding a desired polypeptide or nucleic acid.
(3) Optionally, a nucleic acid comprising a regulatory polynucleotide of SEQ ID No. 486, or the sequence complementary thereto or a biologically active fragment thereof.
i) a DNA construct as described previously in the present specification.
The transgenic animals of the invention thus contain specific sequences of exogenous genetic material such as the nucleotide sequences described above in detail.
In a first preferred embodiment, these transgenic animals may be good experimental models in order to study the diverse pathologies related to cell differentiation, in particular concerning the transgenic animals within the genome of which has been inserted one or several copies of a polynucleotide encoding a native MGST-II protein, or alternatively a mutant MGST-II protein.
In a second preferred embodiment, these transgenic animals may express a desired polypeptide of interest under the control of the regulatory polynucleotides of the MGST-II gene, leading to good yields in the synthesis of this protein of interest, and eventually a tissue specific expression of this protein of interest.
The design of the transgenic animals of the invention may be made according to the conventional techniques well known from the one skilled in the art. For more details regarding the production of transgenic animals, and specifically transgenic mice, it may be referred to U.S. Pat. Nos. 4,873,191, issued Oct. 10, 1989, U.S. Pat. No. 5,464,764 issued Nov. 7, 1995 and U.S. Pat. No. 5,789,215, issued Aug. 4, 1998, these documents being herein incorporated by reference to disclose methods producing transgenic mice.
Transgenic animals of the present invention are produced by the application of procedures which result in an animal with a genome that has incorporated exogenous genetic material. The procedure involves obtaining the genetic material, or a portion thereof, which encodes either a MGST-II coding sequence, a MGST-II regulatory polynucleotide or a DNA sequence encoding a MGST-II antisense polynucleotide such as described in the present specification.
A recombinant polynucleotide of the invention is inserted into an embryonic or ES stem cell line. The insertion is preferably made using electroporation, such as described by Thomas et al. (Cell 51:503-512, 1987). The cells subjected to electroporation are screened (e.g. by selection via selectable markers, by PCR or by Southern blot analysis) to find positive cells which have integrated the exogenous recombinant polynucleotide into their genome, preferably via an homologous recombination event. An illustrative positive-negative selection procedure that may be used according to the invention is described by Mansour et al. (Nature 336:348-352, 1988).
Then, the positive cells are isolated, cloned and injected into 3.5 days old blastocysts from mice, such as described by Bradley (xe2x80x9cProduction and Analysis of Chimaeric Mice,xe2x80x9d E. J. Robertson (Ed), Teratocarcinomas and embryonic stem cells: A practical approach IRL Press, Oxford, 113, 1987). The blastocysts are then inserted into a female host animal and allowed to grow to term.
Alternatively, the positive ES cells are brought into contact with embryos at the 2.5 days old 8-16 cell stage (morulae) such as described by Wood et al. (Proc. Natl. Acad. Sci. U.S.A. 90:4582-4585, 1993) or by Nagy et al. (Proc. Natl. Acad. Sci. USA. 90: 8424-8428, 1993), the ES cells being internalized to colonize extensively the blastocyst including the cells which will give rise to the germ line.
The offspring of the female host are tested to determine which animals are transgenic e.g. include the inserted exogenous DNA sequence and which are wild-type. Thus, the present invention also concerns a transgenic animal containing a nucleic acid, a recombinant expression vector or a recombinant host cell according to the invention.
A further object of the invention consists of recombinant host cells obtained from a transgenic animal described herein.
Recombinant cell lines may be established in vitro from cells obtained from any tissue of a transgenic animal according to the invention, for example by transfection of primary cell cultures with vectors expressing onc-genes such as SV40 large T antigen, as described by Chou (Mol. Endocrinol. 3:1511-1514, 1989) and Shay et al. (Biochem. Biophys. Acta. 1072:1-7, 1991).
E. MGST-II Polypeptides
The term xe2x80x9cMGST-II polypeptidesxe2x80x9d is used herein to embrace all of the proteins and polypeptides of the present invention. Also forming part of the invention are polypeptides encoded by the polynucleotides of the invention, as well as fusion polypeptides comprising such polypeptides. The invention embodies MGST-II proteins from humans, including isolated or purified MGST-II proteins consisting, consisting essentially, or comprising the sequence of SEQ ID Nos. 488 and 489. It should be noted the MGST-II proteins of the invention are based on the naturally-occurring variants of the amino acid sequence of human MGST-II.
In a first embodiment, the present invention provides a variant MGST-II protein; wherein the Tyr residue of amino acid position 93 has been replaced with a His residue. Variant proteins and the fragments thereof which contain amino acid position 93 are collectively referred to herein as xe2x80x9c93-His variants.xe2x80x9d More particularly, the present invention embodies isolated, purified, and recombinant polypeptides comprising a contiguous span of at least 6 amino acids, preferably at least 8 to 10 amino acids, more preferably at least 12, 15, 20, 25, 30, 40, 50, or 100 amino acids of SEQ ID No. 488, wherein said contiguous span comprises a His residue at amino acid position 93. In this amino acid substitution the original residue (Tyr) is replaced by a non-equivalent amino acid (His) presenting different chemical properties.
The present invention further provides another naturally-occurring variant of the MGST-II protein that consists or consists essentially of amino acids 1-109 of SEQ ID No. 488. This variant MGST-II polypeptide corresponds to one allele of biallelic marker 10-290-37.
Another naturally-occurring variant of the MGST-II protein of the present invention is encoded by a cDNA obtained by alternative splicing. MGST-II cDNAs and cDNA variants are further described above. This variant polypeptide of a sequence from SEQ ID No. 489 is identical to the MGST-II protein of SEQ ID No. 488 from amino acid position 1 to amino acid position 19 but comprises 11 additional amino acids. The present invention embodies isolated, purified, and recombinant polypeptides comprising, consisting of or consisting essentially of an amino acid sequence from SEQ ID No. 489. Moreover, the present invention embodies isolated, purified, and recombinant polypeptides comprising a contiguous span of at least 6 amino acids, preferably at least 8 to 10 amino acids, more preferably at least 12, 15, 20, 25, or 30 amino acids of SEQ ID No. 489, wherein said contiguous span comprises a least one of amino acid positions 20 to 30 of SEQ ID No. 489.
All the variant MGST-II polypeptides described above most probably show alterations in the activity, specificity and function of the MGST-II enzyme. In preferred embodiments the polypeptides of the present invention comprise the site of a mutation or functional mutation, including a deletion, substitution or truncation in the amino acid sequence in the MGST-II protein.
MGST-II proteins are preferably isolated from human or mammalian tissue samples or expressed from human or mammalian genes. The MGST-II polypeptides of the invention can be made using routine expression methods known in the art. The polynucleotide encoding the desired polypeptide, is ligated into an expression vector suitable for any convenient host. Both eukaryotic and prokaryotic host systems are used in forming recombinant polypeptides. The polypeptide is then isolated from lysed cells or from the culture medium and purified to the extent needed for its intended use. Purification is by any technique known in the art, for example, differential extraction, salt fractionation, chromatography, centrifugation, and the like. See, for example, Methods in Enzymology for a variety of methods for purifying proteins.
In addition, shorter protein fragments are produced by chemical synthesis. Alternatively the proteins of the invention are extracted from cells or tissues of humans or non-human animals. Methods for purifying proteins are known in the art, and include the use of detergents or chaotropic agents to disrupt particles followed by differential extraction and separation of the polypeptides by ion exchange chromatography, affinity chromatography, sedimentation according to density, and gel electrophoresis.
Any MGST-II cDNA of the invention is used to express MGST-II proteins and polypeptides. The nucleic acid encoding the MGST-II protein or polypeptide to be expressed is operably linked to a promoter in an expression vector using conventional cloning technology. The MGST-II insert in the expression vector may comprise the full coding sequence for the MGST-II protein or a portion thereof. For example, the MGST-II derived insert may encode a polypeptide comprising a contiguous span of at least 6 amino acids, preferably at least 8 to 10 amino acids, more preferably at least 12, 15, 20, 25, 30, 40, 50, or 100 amino acids of SEQ ID No. 488, wherein said contiguous span comprises a His residue at amino acid position 93. The MGST-II derived insert may further encode a polypeptide comprising, consisting of or consisting essentially of an amino acid sequence from amino acid positions 1-108 of SEQ ID No. 488. The MGST-II derived insert may further encode a polypeptide comprising, consisting of or consisting essentially of an amino acid sequence from SEQ ID No. 489. The MGST-II derived insert may also encode a polypeptide comprising a contiguous span of at least 6 amino acids, preferably at least 8 to 10 amino acids, more preferably at least 12, 15, 20, 25,or 30 amino acids of SEQ ID No. 489, wherein said contiguous span comprises a least one of amino acid positions 20 to 30 of SEQ ID No. 489.
The expression vector is any of the mammalian, yeast, insect or bacterial expression systems known in the art. Commercially available vectors and expression systems are available from a variety of suppliers including Genetics Institute (Cambridge, Mass.), Stratagene (La Jolla, Calif.), Promega (Madison, Wis.), and Invitrogen (San Diego, Calif.). If desired, to enhance expression and facilitate proper protein folding, the codon context and codon pairing of the sequence is optimized for the particular expression organism in which the expression vector is introduced, as explained by Hatfield, et al., U.S. Pat. No. 5,082,767.
In one embodiment, the entire coding sequence of the MGST-II cDNA through the poly A signal of the cDNA is operably linked to a promoter in the expression vector. Alternatively, if the nucleic acid encoding a portion of the MGST-II protein lacks a methionine to serve as the initiation site, an initiating methionine can be introduced next to the first codon of the nucleic acid using conventional techniques. Similarly, if the insert from the MGST-II cDNA lacks a poly A signal, this sequence can be added to the construct by, for example, splicing out the Poly A signal from pSG5 (Stratagene) using BglI and SalI restriction endonuclease enzymes and incorporating it into the mammalian expression vector pXT1 (Stratagene). pXT1 contains the LTRs and a portion of the gag gene from Moloney Murine Leukemia Virus. The position of the LTRs in the construct allows efficient stable transfection. The vector includes the Herpes Simplex Thymidine Kinase promoter and the selectable neomycin gene. The nucleic acid encoding the MGST-II protein or a portion thereof is obtained by PCR from a bacterial vector containing a MGST-II cDNA of the present invention using oligonucleotide primers complementary to the MGST-II cDNA or portion thereof and containing restriction endonuclease sequences for Pst I incorporated into the 5xe2x80x2 primer and Bgl II at the 5xe2x80x2 end of the corresponding cDNA 3xe2x80x2 primer, taking care to ensure that the sequence encoding the MGST-II protein or a portion thereof is positioned properly with respect to the poly A signal. The purified fragment obtained from the resulting PCR reaction is digested with PstI, blunt ended with an exonuclease, digested with Bgl II, purified and ligated to pXT1, now containing a poly A signal and digested with BglII.
The ligated product is transfected into mouse NIH 3T3 cells using Lipofectin (Life Technologies, Inc., Grand Island, N.Y.) under conditions outlined in the product specification. Positive transfectants are selected after growing the transfected cells in 600 ug/ml G418 (Sigma, St. Louis, Mo.).
Alternatively, the nucleic acids encoding the MGST-II protein or a portion thereof is cloned into pED6dpc2 (Genetics Institute, Cambridge, Mass.). The resulting pED6dpc2 constructs is transfected into a suitable host cell, such as COS 1 cells. Methotrexate resistant cells are selected and expanded.
The above procedures may also be used to express a mutant MGST-II protein responsible for a detectable phenotype or a portion thereof.
The expressed proteins are purified using conventional purification techniques such as ammonium sulfate precipitation or chromatographic separation based on size or charge. The protein encoded by the nucleic acid insert may also be purified using standard immunochromatography techniques. In such procedures, a solution containing the expressed MGST-II protein or portion thereof, such as a cell extract, is applied to a column having antibodies against the MGST-II protein or portion thereof is attached to the chromatography matrix. The expressed protein is allowed to bind the immunochromatography column. Thereafter, the column is washed to remove non-specifically bound proteins. The specifically bound expressed protein is then released from the column and recovered using standard techniques.
To confirm expression of the MGST-II protein or a portion thereof, the proteins expressed from host cells containing an expression vector containing an insert encoding the MGST-II protein or a portion thereof can be compared to the proteins expressed in host cells containing the expression vector without an insert. The presence of a band in samples from cells containing the expression vector with an insert which is absent in samples from cells containing the expression vector without an insert indicates that the MGST-II protein or a portion thereof is being expressed. Generally, the band will have the mobility expected for the MGST-II protein or portion thereof. However, the band may have a mobility different than that expected as a result of modifications such as glycosylation, ubiquitination, or enzymatic cleavage.
Antibodies capable of specifically recognizing the expressed MGST-II protein or a portion thereof, are described below.
If antibody production is not possible, the nucleic acids encoding the MGST-II protein or a portion thereof is incorporated into expression vectors designed for use in purification schemes employing chimeric polypeptides. In such strategies the nucleic acid encoding the MGST-II protein or a portion thereof is inserted in frame with the gene encoding the other half of the chimera. The other half of the chimera is xcex2-globin or a nickel binding polypeptide encoding sequence. A chromatography matrix having antibody to xcex2-globin or nickel attached thereto is then used to purify the chimeric protein. Protease cleavage sites is engineered between the xcex2-globin gene or the nickel binding polypeptide and the MGST-II protein or portion thereof. Thus, the two polypeptides of the chimera are separated from one another by protease digestion.
One useful expression vector for generating xcex2-globin chimerics is pSG5 (Stratagene), which encodes rabbit xcex2-globin. Intron II of the rabbit xcex2-globin gene facilitates splicing of the expressed transcript, and the polyadenylation signal incorporated into the construct increases the level of expression. These techniques are well known to those skilled in the art of molecular biology. Standard methods are published in methods texts such as Davis et al., (Basic Methods in Molecular Biology, L. G. Davis, M. D. Dibner, and J. F. Battey, ed., Elsevier Press, NY, 1986) and many of the methods are available from Stratagene, Life Technologies, Inc., or Promega. Polypeptide may additionally be produced from the construct using in vitro translation systems such as the In vitro Express(trademark) Translation Kit (Stratagene).
F. Production of Antibodies Against MGST-II Polypeptides
Any MGST-II polypeptide or whole protein may be used to generate antibodies capable of specifically binding to expressed MGST-II protein or fragments thereof or variants thereof. Preferably the antibody compositions of the invention are capable of specifically binding to the 93-His variant of the MGST-II protein. Alternatively the antibody compositions of the present invention are capable of specifically binding the variant MGST-II polypeptide of SEQ ID No. 489. A preferred embodiment of the invention encompasses isolated or purified antibody compositions capable of selectively binding, or which are capable of binding to an epitope-containing fragment of a polypeptide of the invention, wherein said epitope comprises at least one amino acid position selected from the group consisting of His residue at amino acid position 93 of SEQ ID No. 488 and of amino acid positions 20-30 of SEQ ID No. 489. For an antibody composition to specifically bind to these MGST-II variants it must demonstrate at least a 5%, 10%, 15%, 20%, 25%, 50%, or 100% greater binding affinity for full length MGST-II variants in an ELISA, RIA, or other antibody-based binding assay than to full length MGST-II protein described in SEQ ID No. 488. Affinity of the antibody composition for the epitope can further be determined by preparing competitive binding curves, as described, for example, by Fisher, D. (Chap. 42 in: Manual of Clinical Immunology, 2d Ed. (Rose and Friedman,Eds.) Amer. Soc. For Microbiol., Washington, D.C., 1980).
The present invention also contemplates the use of variant MGST-II polypeptides in the manufacture of antibodies. In a preferred embodiment such polypeptides are useful in the manufacture of antibodies to detect the presence and absence of the 93-His variant and of the MGST-II variant of SEQ ID No. 489.
Non-human animals or mammals, whether wild-type or transgenic, which express a different species of MGST-II than the one to which antibody binding is desired, and animals which do not express MGST-II (i.e. an MGST-II knock out animal as described in herein) are particularly useful for preparing antibodies. MGST-II knock out animals will recognize all or most of the exposed regions of MGST-II as foreign antigens, and therefore produce antibodies with a wider array of MGST-II epitopes. Moreover, smaller polypeptides with only 10 to 30 amino acids may be useful in obtaining specific binding to the 93-His variant and to the MGST-II variant of SEQ ID No. 489. In addition, the humoral immune system of animals which produce a species of MGST-II that resembles the antigenic sequence will preferentially recognize the differences between the animal""s native MGST-II species and the antigen sequence, and produce antibodies to these unique sites in the antigen sequence. Such a technique will be particularly useful in obtaining antibodies that specifically bind to the 93-His variant and to the MGST-II variant of SEQ ID No. 489. The preparation of antibody compositions is further described in Example 6.
Antibody preparations prepared according to the present invention are useful in quantitative immunoassays which determine concentrations of antigen-bearing substances in biological samples; they are also used semi-quantitatively or qualitatively to identify the presence of antigen in a biological sample. The antibodies may also be used in therapeutic compositions for killing cells expressing the protein or reducing the levels of the protein in the body. The antibodies of the invention may be labeled, either by a radioactive, a fluorescent or an enzymatic label. Consequently, the invention is also directed to a method for detecting specifically the presence of a variant MGST-II polypeptide according to the invention in a biological sample, said method comprising the following steps: a) bringing into contact the biological sample with a polyclonal or monoclonal antibody that specifically binds a variant MGST-II polypeptide or to a peptide fragment or variant thereof; and b) detecting the antigen-antibody complex formed. The invention also concerns a diagnostic kit for detecting in vitro the presence of a variant MGST-II polypeptide according to the present invention in a biological sample, wherein said kit comprises: a) a polyclonal or monoclonal antibody that specifically binds a variant MGST-II polypeptide or to a peptide fragment or variant thereof, optionally labeled; b) a reagent allowing the detection of the antigen-antibody complexes formed, said reagent carrying optionally a label, or being able to be recognized itself by a labeled reagent, more particularly in the case when the above-mentioned monoclonal or polyclonal antibody is not labeled by itself.
II. Methods for De Novo Identification of Biallelic Markers
Large fragments of human DNA, carrying genes of interest involved in the biotransformation of xenobiotics such as therapeutic drugs; were cloned, sequenced and screened for biallelic markers. Biallelic markers within the candidate genes themselves as well as markers located on the same genomic fragment were identified. It will be clear to one of skill in the art that large fragments of human genomic DNA may be obtained from any appropriate source and may be cloned into a number of suitable vectors.
In a preferred embodiment of the invention, BAC (Bacterial Artificial Chromosomes) vectors were used to construct DNA libraries covering the entire human genome. Specific amplification primers were designed for each candidate gene and the BAC library was screened by PCR until there was at least one positive BAC clone per candidate gene. Genomic sequence, screened for biallelic markers, was generated by sequencing ends of BAC subclones. Details of a preferred embodiment are provided in Example 1. As a preferred alternative to sequencing the ends of an adequate number of BAC subclones, high throughput deletion-based sequencing vectors, which allow the generation of a high quality sequence information covering fragments of about 6 kb, may be used. Having sequence fragments longer than 2.5 or 3 kb enhances the chances of identifying biallelic markers therein. Methods of constructing and sequencing a nested set of deletions are disclosed in the related U.S. patent application entitled xe2x80x9cHigh Throughput DNA Sequencing Vectorxe2x80x9d (Ser. No. 09/058,746).
In another embodiment of the invention, genomic sequences of candidate genes were available in public databases allowing direct screening for biallelic markers.
Any of a variety of methods can be used to screen a genomic fragment for single nucleotide polymorphisms such as differential hybridization with oligonucleotide probes, detection of changes in the mobility measured by gel electrophoresis or direct sequencing of the amplified nucleic acid. A preferred method for identifying biallelic markers involves comparative sequencing of genomic DNA fragments from an appropriate number of unrelated individuals.
In a first embodiment, DNA samples from unrelated individuals are pooled together, following which the genomic DNA of interest is amplified and sequenced. The nucleotide sequences thus obtained are then analyzed to identify significant polymorphisms. One of the major advantages of this method resides in the fact that the pooling of the DNA samples substantially reduces the number of DNA amplification reactions and sequencing reactions, which must be carried out. Moreover, this method is sufficiently sensitive so that a biallelic marker obtained thereby usually demonstrates a sufficient frequency of its less common allele to be useful in conducting association studies. Usually, the frequency of the least common allele of a biallelic marker identified by this method is at least 10%.
In a second embodiment, the DNA samples are not pooled and are therefore amplified and sequenced individually. This method is usually preferred when biallelic markers need to be identified in order to perform association studies within candidate genes. Preferably, highly relevant gene regions such as promoter regions or exon regions may be screened for biallelic markers. A biallelic marker obtained using this method may show a lower degree of informativeness for conducting association studies, e.g. if the frequency of its less frequent allele may be less than about 10%. Such a biallelic marker will however be sufficiently informative to conduct association studies and it will further be appreciated that including less informative biallelic markers in the genetic analysis studies of the present invention, may allow in some cases the direct identification of causal mutations, which may, depending on their penetrance, be rare mutations.
The following is a description of the various parameters of a preferred method used by the inventors for the identification of the biallelic markers of the present invention.
A. Genomic DNA Samples
The genomic DNA samples from which the biallelic markers of the present invention are generated are preferably obtained from unrelated individuals corresponding to a heterogeneous population of known ethnic background. The number of individuals from whom DNA samples are obtained can vary substantially, preferably from about 10 to about 1000, more preferably from about 50 to about 200 individuals. Usually, DNA samples are collected from at least about 100 individuals in order to have sufficient polymorphic diversity in a given population to identify as many markers as possible and to generate statistically significant results.
As for the source of the genomic DNA to be subjected to analysis, any test sample can be foreseen without any particular limitation. These test samples include biological samples, which can be tested by the methods of the present invention described herein, and include human and animal body fluids such as whole blood, serum, plasma, cerebrospinal fluid, urine, lymph fluids, and various external secretions of the respiratory, intestinal and genitourinary tracts, tears, saliva, milk, white blood cells, myelomas and the like; biological fluids such as cell culture supernatants; fixed tissue specimens including tumor and non-tumor tissue and lymph node tissues; bone marrow aspirates and fixed cell specimens. The preferred source of genomic DNA used in the present invention is from peripheral venous blood of each donor. Techniques to prepare genomic DNA from biological samples are well known to the skilled technician. Details of a preferred embodiment are provided in Example 1. The person skilled in the art can choose to amplify pooled or unpooled DNA samples.
B. DNA Amplification
The identification of biallelic markers in a sample of genomic DNA may be facilitated through the use of DNA amplification methods. DNA samples can be pooled or unpooled for the amplification step. DNA amplification techniques are well known to those skilled in the art. Various methods to amplify DNA fragments carrying biallelic markers are further described hereinafter in III.B. The PCR technology is the preferred amplification technique used to identify new biallelic markers.
In a first embodiment, biallelic markers are identified using genomic sequence information generated by the inventors. Genomic DNA fragments, such as the inserts of the BAC clones described above, are sequenced and used to design primers for the amplification of 500 bp fragments. These 500 bp fragments are amplified from genomic DNA and are scanned for biallelic markers. Primers may be designed using the OSP software (Hillier L. and Green P., 1991), the disclosures of which are incorporated herein by reference in their entirety. All primers may contain, upstream of the specific target bases, a common oligonucleotide tail that serves as a sequencing primer. Those skilled in the art are familiar with primer extensions, which can be used for these purposes.
In another embodiment of the invention, genomic sequences of candidate genes are available in public databases allowing direct screening for biallelic markers. Preferred primers, useful for the amplification of genomic sequences encoding the candidate genes, focus on promoters, exons and splice sites of the genes. A biallelic marker present in these functional regions of the gene have a higher probability to be a causal mutation.
Preferred primers include those disclosed in Table 17.
C. Sequencing of Amplified Genomic DNA and Identification of Single Nucleotide Polymophisms
The amplification products generated as described above, are then sequenced using any method known and available to the skilled technician. Methods for sequencing DNA using either the dideoxy-mediated method (Sanger method) or the Maxam-Gilbert method are widely known to those of ordinary skill in the art. Such methods are for example disclosed in Maniatis et al. (Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Second Edition, 1989), the disclosure of which is incorporated herein by reference in its entirety. Alternative approaches include hybridization to high-density DNA probe arrays as described in Chee et al. (Science 274, 610, 1996), the disclosure of which is incorporated herein by reference in its entirety.
Preferably, the amplified DNA is subjected to automated dideoxy terminator sequencing reactions using a dye-primer cycle sequencing protocol. The products of the sequencing reactions are run on sequencing gels and the sequences are determined using gel image analysis. The polymorphism search is based on the presence of superimposed peaks in the electrophoresis pattern resulting from different bases occurring at the same position. Because each dideoxy terminator is labeled with a different fluorescent molecule, the two peaks corresponding to a biallelic site present distinct colors corresponding to two different nucleotides at the same position on the sequence. However, the presence of two peaks can be an artifact due to background noise. To exclude such an artifact, the two DNA strands are sequenced and a comparison between the peaks is carried out. In order to be registered as a polymorphic sequence, the polymorphism has to be detected on, both strands.
The above procedure permits those amplification products, which contain biallelic markers to be identified. The detection limit for the frequency of biallelic polymorphisms detected by sequencing pools of 100 individuals is approximately 0.1 for the minor allele, as verified by sequencing pools of known allelic frequencies. However, more than 90% of the biallelic polymorphisms detected by the pooling method have a frequency for the minor allele higher than 0.25. Therefore, the biallelic markers selected by this method have a frequency of at least 0.1 for the minor allele and less than 0.9 for the major allele. Preferably at least 0.2 for the minor allele and less than 0.8 for the major allele, more preferably at least 0.3 for the minor allele 30 and less than 0.7 for the major allele, thus a heterozygosity rate higher than 0.18, preferably higher than 0.32, more preferably higher than 0.42.
In another embodiment, biallelic markers are detected by sequencing individual DNA samples, the frequency of the minor allele of such a biallelic marker may be less than 0.1.
The markers carried by the same fragment of genomic DNA, such as the insert in a BAC clone, need not necessarily be ordered with respect to one another within the genomic fragment to conduct association studies. However, in some embodiments of the present invention, the order of biallelic markers carried by the same fragment of genomic DNA are determined.
D. Validation of the Biallelic Markers of the Present Invention
The polymorphisms are evaluated for their usefulness as genetic markers by validating that both alleles are present in a population. Validation of the biallelic markers is accomplished by genotyping a group of individuals by a method of the invention and demonstrating that both alleles are present. Microsequencing is a preferred method of genotyping alleles. The validation by genotyping step may be performed on individual samples derived from each individual in the group or by genotyping a pooled sample derived from more than one individual. The group can be as small as one individual if that individual is heterozygous for the allele in question. Preferably the group contains at least three individuals, more preferably the group contains five or six individuals, so that a single validation test will be more likely to result in the validation of more of the biallelic markers that are being tested. It should be noted, however, that when the validation test is performed on a small group it may result in a false negative result if as a result of sampling error none of the individuals tested carries one of the two alleles. Thus, the validation process is less useful in demonstrating that a particular initial result is an artifact, than it is at demonstrating that there is a bonafide biallelic marker at a particular position in a sequence. For an indication of whether a particular biallelic marker has been validated see Table 11(A-B). All of the genotyping, haplotyping, association, and interaction study methods of the invention may optionally be performed solely with validated biallelic markers.
E. Evaluation of the Frequency of the Biallelic Markers of the Present Invention
The validated biallelic markers are further evaluated for their usefulness as genetic markers by determining the frequency of the least common allele at the biallelic marker site. The determination of the least common allele is accomplished by genotyping a group of individuals by a method of the invention and demonstrating that both alleles are present. This determination of frequency by genotyping step may be performed on individual samples derived from each individual in the group or by genotyping a pooled sample derived from more than one individual. The group must be large enough to be representative of the population as a whole. Preferably the group contains at least 20 individuals, more preferably the group contains at least 50 individuals, most preferably the group contains at least 100 individuals. Of course the larger the group the greater the accuracy of the frequency determination because of reduced sampling error. For an indication of the frequency for the less common allele of a particular biallelic marker of the invention see Table 11(A-B). A biallelic marker wherein the frequency of the less common allele is 30% or more is termed a xe2x80x9chigh quality biallelic marker.xe2x80x9d All of the genotyping, haplotyping, association, and interaction study methods of the invention may optionally be performed solely with high quality biallelic markers.
III. Methods of Genotyping an Individual for Biallelic Markers
Methods are provided to genotype a biological sample for one or more biallelic markers of the present invention, all of which may be performed in vitro. Such methods of genotyping comprise determining the identity of a nucleotide at a DME-related biallelic marker by any method known in the art. These methods find use in genotyping case-control populations in association studies as well as individuals in the context of detection of alleles of biallelic markers which, are known to be associated with a given trait, in which case both copies of the biallelic marker present in individual""s genome are determined so that an individual may be classified as homozygous or heterozygous for a particular allele.
These genotyping methods can be performed nucleic acid samples derived from a single individual or pooled DNA samples.
Genotyping can be performed using similar methods as those described above for the identification of the biallelic markers, or using other genotyping methods such as those further described below. In preferred embodiments, the comparison of sequences of amplified genomic fragments from different individuals is used to identify new biallelic markers whereas microsequencing is used for genotyping known biallelic markers in diagnostic and association study applications.
A. Source of DNA for Genotyping
Any source of nucleic acids, in purified or non-purified form, can be utilized as the starting nucleic acid, provided it contains or is suspected of containing the specific nucleic acid sequence desired. DNA or RNA may be extracted from cells, tissues, body fluids and the like as described above in II.A. xe2x80x9cGenomic DNA Samples.xe2x80x9d While nucleic acids for use in the genotyping methods of the invention can be derived from any mammalian source, the test subjects and individuals from which nucleic acid samples are taken are generally understood to be human.
B. Amplification of DNA Fragments Comprising Biallelic Markers
Methods and polynucleotides are provided to amplify a segment of nucleotides comprising one or more biallelic marker of the present invention. It will be appreciated that amplification of DNA fragments comprising biallelic markers may be used in various methods and for various purposes and is not restricted to genotyping. Nevertheless, many genotyping methods, although not all, require the previous amplification of the DNA region carrying the biallelic marker of interest. Such methods specifically increase the concentration or total number of sequences that span the biallelic marker or include that site and sequences located either distal or proximal to it. Diagnostic assays may also rely on amplification of DNA segments carrying a biallelic marker of the present invention.
Amplification of DNA may be achieved by any method known in the art. The established PCR (polymerase chain reaction) method or by developments thereof or alternatives. Amplification methods which can be utilized herein include but are not limited to Ligase Chain Reaction (LCR) as described in EP A 320 308 and EP A 439 182, Gap LCR (Wolcott, M. J., Clin. Mcrobiol. Rev. 5:370-386), the so-called xe2x80x9cNASBAxe2x80x9d or xe2x80x9c3SRxe2x80x9d technique described in Guatelli J. C. et al. (Proc. Natl. Acad. Sci. USA 87:1874-1878, 1990) and in Compton J. (Nature 350:91-92, 1991), Q-beta amplification as described in European Patent Application no 4544610, strand displacement amplification as described in Walker et al. (Clin. Chem. 42:9-13, 1996) and EP A 684 315 and, target mediated amplification as described in PCT Publication WO 9322461, the disclosures of which are incorporated herein by reference in their entirety.
LCR and Gap LCR are exponential amplification techniques, both depend on DNA ligase to join adjacent primers annealed to a DNA molecule. In Ligase Chain Reaction (LCR), probe pairs are used which include two primary (first and second) and two secondary (third and fourth) probes, all of which are employed in molar excess to target. The first probe hybridizes to a first segment of the target strand and the second probe hybridizes to a second segment of the target strand, the first and second segments being contiguous so that the primary probes abut one another in 5xe2x80x2 phosphate-3xe2x80x2hydroxyl relationship, and so that a ligase can covalently fuse or ligate the two probes into a fused product. In addition, a third (secondary) probe can hybridize to a portion of the first probe and a fourth (secondary) probe can hybridize to a portion of the second probe in a similar abutting fashion. Of course, if the target is initially double stranded, the secondary probes also will hybridize to the target complement in the first instance. Once the ligated strand of primary probes is separated from the target strand, it will hybridize with the third and fourth probes which can be ligated to form a complementary, secondary ligated product. It is important to realize that the ligated products are functionally equivalent to either the target or its complement. By repeated cycles of hybridization and ligation, amplification of the target sequence is achieved. A method for multiplex LCR has also been described (WO 9320227), the disclosure of which is incorporated herein by reference in its entirety. Gap LCR (GLCR) is a version of LCR where the probes are not adjacent but are separated by 2 to 3 bases.
For amplification of mRNAs, it is within the scope of the present invention to reverse transcribe mRNA into cDNA followed by polymerase chain reaction (RT-PCR); or, to use a single enzyme for both steps as described in U.S. Pat. No. 5,322,770 or, to use Asymmetric Gap LCR (RT-AGLCR) as described by Marshall R. L. et al. (PCR Methods and Applications 4:80-84, 1994), the disclosures of which are incorporated herein by reference in their entirety. AGLCR is a modification of GLCR that allows the amplification of RNA.
Some of these amplification methods are particularly suited for the detection of single nucleotide polymorphisms and allow the simultaneous amplification of a target sequence and the identification of the polymorphic nucleotide as it is further described in xe2x80x9cIIIC. Methods of Genotyping DNA samples for Biallelic Markers.xe2x80x9d
The PCR technology is the preferred amplification technique used in the present invention. A variety of PCR techniques are familiar to those skilled in the art. For a review of PCR technology, see Molecular Cloning to Genetic Engineering White, B. A. Ed. in Methods in Molecular Biology 67: Humana Press, Totowa (1997) and the publication entitled xe2x80x9cPCR Methods and Applicationsxe2x80x9d (1991, Cold Spring Harbor Laboratory Press), the disclosure of which is incorporated herein by reference in its entirety. In each of these PCR procedures, PCR primers on either side of the nucleic acid sequences to be amplified are added to a suitably prepared nucleic acid sample along with dNTPs and a thermostable polymerase such as Taq polymerase, Pfu polymerase, or Vent polymerase. The nucleic acid in the sample is denatured and the PCR primers are specifically hybridized to complementary nucleic acid sequences in the sample. The hybridized primers are extended. Thereafter, another cycle of denaturation, hybridization, and extension is initiated. The cycles are repeated multiple times to produce an amplified fragment containing the nucleic acid sequence between the primer sites. PCR has further been described in several patents including U.S. Pat. Nos. 4,683,195, 4,683,202 and 4,965,188, the disclosures of which are incorporated herein by reference in their entirety.
The identification of biallelic markers as described above allows the design of appropriate oligonucleotides, which can be used as primers to amplify DNA fragments comprising the biallelic markers of the present invention. Amplification can be performed using the primers initially used to discover new biallelic markers which are described herein or any set of primers allowing the amplification of a DNA fragment comprising a biallelic marker of the present invention. Primers can be prepared by any suitable method. As for example, direct chemical synthesis by a method such as the phosphodiester method of Narang S. A. et al. (Methods Enzymol. 68:90-98, 1979), the phosphodiester method of Brown E. L. et al. (Methods Enzymol. 68:109-151, 1979), the diethylphosphoramidite method of Beaucage et al. (Tetrahedron Lett. 22:1859-1862, 1981) and the solid support method described in EP 0 707 592, the disclosures of which are incorporated herein by reference in their entirety.
In some embodiments the present invention provides primers for amplifying a DNA fragment containing one or more biallelic markers of the present invention. Preferred amplification primers are listed in Table 17. It will be appreciated that the primers listed are merely exemplary and that any other set of primers which produce amplification products containing one or more biallelic markers of the present invention.
The primers are selected to be substantially complementary to the different strands of each specific sequence to be amplified. The length of the primers of the present invention can range from 8 to 100 nucleotides, preferably from 8 to 50, 8 to 30 or more preferably 8 to 25 nucleotides. Shorter primers tend to lack specificity for a target nucleic acid sequence and generally require cooler temperatures to form sufficiently stable hybrid complexes with the template. Longer primers are expensive to produce and can sometimes self-hybridize to form hairpin structures. The formation of stable hybrids depends on the melting temperature (Tm) of the DNA. The Tm depends on the length of the primer, the ionic strength of the solution and the G+C content. The higher the G+C content of the primer, the higher is the melting temperature because G:C pairs are held by three H bonds whereas A:T pairs have only two. The G+C content of the amplification primers of the present invention preferably ranges between 10 and 75%, more preferably between 35 and 60%, and most preferably between 40 and 55%. The appropriate length for primers under a particular set of assay conditions may be empirically determined by one of skill in the art.
The spacing of the primers determines the length of the segment to be amplified. In the context of the present invention amplified segments carrying biallelic markers can range in size from at least about 25 bp to 35 kbp. Amplification fragments from 25-3000 bp are typical, fragments from 50-1000 bp are preferred and fragments from 100-600 bp are highly preferred. It will be appreciated that amplification primers for the biallelic markers may be any sequence which allow the specific amplification of any DNA fragment carrying the markers. Amplification primers may be labeled or immobilized on a solid support as described in I.
C. Methods of Genotyping DNA samples for Biallelic Markers
Any method known in the art can be used to identify the nucleotide present at a biallelic marker site. Since the biallelic marker allele to be detected has been identified and specified in the present invention, detection will prove simple for one of ordinary skill in the art by employing any of a number of techniques. Many genotyping methods require the previous amplification of the DNA region carrying the biallelic marker of interest. While the amplification of target or signal is often preferred at present, ultrasensitive detection methods which do not require amplification are also encompassed by the present genotyping methods. Methods well-known to those skilled in the art that can be used to detect biallelic polymorphisms include methods such as, conventional dot blot analyzes, single strand conformational polymorphism analysis (SSCP) described by Orita et al. (Proc. Natl. Acad. Sci. U.S.A 86:27776-2770, 1989), denaturing gradient gel electrophoresis (DGGE), heteroduplex analysis, mismatch cleavage detection, and other conventional techniques as described in Sheffield, V. C. et al. (Proc. Natl. Acad. Sci. USA 49:699-706, 1991), White et al. (Genomics 12:301-306, 1992), Grompe, M. et al. (Proc. Natl. Acad. Sci. USA 86:5855-5892, 1989) and Grompe, M. (Nature Genetics 5:111-117, 1993), the disclosures of which are incorporated herein by reference in their entirety. Another method for determining the identity of the nucleotide present at a particular polymorphic site employs a specialized exonuclease-resistant nucleotide derivative as described in U.S. Pat. No. 4,656,127, the disclosure of which is incorporated herein by reference in its entirety.
Preferred methods involve directly determining the identity of the nucleotide present at a biallelic marker site by sequencing assay, enzyme-based mismatch detection assay, or hybridization assay. The following is a description of some preferred methods. A highly preferred method is the microsequencing technique. The term xe2x80x9csequencing assayxe2x80x9d is used herein to refer to polymerase extension of duplex primer/template complexes and includes both traditional sequencing and microsequencing.
Sequencing Assays
The nucleotide present at a polymorphic site can be determined by sequencing methods. In a preferred embodiment, DNA samples are subjected to PCR amplification before sequencing as described above. DNA sequencing methods are described in IIC.
Preferably, the amplified DNA is subjected to automated dideoxy terminator sequencing reactions using a dye-primer cycle sequencing protocol. Sequence analysis allows the identification of the base present at the biallelic marker site.
Microsequencing Assays
In microsequencing methods, a nucleotide at the polymorphic site that is unique to one of the alleles in a target DNA is detected by a single nucleotide primer extension reaction. This method involves appropriate microsequencing primers which, hybridize just upstream of a polymorphic base of interest in the target nucleic acid. A polymerase is used to specifically extend the 3xe2x80x2 end of the primer with one single ddNTP (chain terminator) complementary to the selected nucleotide at the polymorphic site. Next the identity of the incorporated nucleotide is determined in any suitable way.
Typically, microsequencing reactions are carried out using fluorescent ddNTPs and the extended microsequencing primers are analyzed by electrophoresis on ABI 377 sequencing machines to determine the identity of the incorporated nucleotide as described in EP 412 883. Alternatively capillary electrophoresis can be used in order to process a higher number of assays simultaneously. An example of a typical microsequencing procedure that can be used in the context of the present invention is provided in Example 2.
Different approaches can be used to detect the nucleotide added to the microsequencing primer. A homogeneous phase detection method based on fluorescence resonance energy transfer has been described by Chen and Kwok (Nucleic Acids Research 25:347-353 1997) and Chen et al. (Proc. Natl. Acad. Sci. USA 94/20 10756-10761,1997), the disclosures of which are incorporated herein by reference in their entirety. In this method amplified genomic DNA fragments containing polymorphic sites are incubated with a 5xe2x80x2-fluorescein-labeled primer in the presence of allelic dyelabeled dideoxyribonucleoside triphosphates and a modified Taq polymerase. The dye-labeled primer is extended one base by the dye-terminator specific for the allele present on the template. At the end of the genotyping reaction, the fluorescence intensities of the two dyes in the reaction mixture are analyzed directly without separation or purification. All these steps can be performed in the same tube and the fluorescence changes can be monitored in real time. Alternatively, the extended primer may be analyzed by MALDI-TOF Mass Spectrometry. The base at the polymorphic site is identified by the mass added onto the microsequencing primer (see Haff L. A. and Smirnov I. P., Genome Research, 7:378-388, 1997), the disclosures of which are incorporated herein by reference in their entirety.
Microsequencing may be achieved by the established microsequencing method or by developments or derivatives thereof. Alternative methods include several solid-phase microsequencing techniques. The basic microsequencing protocol is the same as described previously, except that the method is conducted as a heterogenous phase assay, in which the primer or the target molecule is immobilized or captured onto a solid support. To simplify the primer separation and the terminal nucleotide addition analysis, oligonucleotides are attached to solid supports or are modified in such ways that permit affinity separation as well as polymerase extension. The 5xe2x80x2 ends and internal nucleotides of synthetic oligonucleotides can be modified in a number of different ways to permit different affinity separation approaches, e.g., biotinylation. If a single affinity group is used on the oligonucleotides, the oligonucleotides can be separated from the incorporated terminator regent. This eliminates the need of physical or size separation. More than one oligonucleotide can be separated from the terminator reagent and analyzed simultaneously if more than one affinity group is used. This permits the analysis of several nucleic acid species or more nucleic acid sequence information per extension reaction. The affinity group need not be on the priming oligonucleotide but could alternatively be present on the template. For example, immobilization can be carried out via an interaction between biotinylated DNA and streptavidin-coated microtitration wells or avidin-coated polystyrene particles. In the same manner oligonucleotides or templates may be attached to a solid support in a high-density format. In such solid phase microsequencing reactions, incorporated ddNTPs can be radiolabeled (Syvxc3xa4nen, Clinica Chimica Acta 226:225-236, 1994) or linked to fluorescein (Livak and Hainer, Human Mutation 3:379-385,1994), the disclosures of which are incorporated herein by reference in their entirety. The detection of radiolabeled ddNTPs can be achieved through scintillation-based techniques. The detection of fluorescein-linked ddNTPs can be based on the binding of antifluorescein antibody conjugated with alkaline phosphatase, followed by incubation with a chromogenic substrate (such as p-nitrophenyl phosphate). Other possible reporter-detection pairs include: ddNTP linked to dinitrophenyl (DNP) and anti-DNP alkaline phosphatase conjugate (Harju et al., Clin. Chem. 39/11 2282-2287, 1993) or biotinylated ddNTP and horseradish peroxidase-conjugated streptavidin with o-phenylenediamine as a substrate (WO 92/15712), the disclosures of which are incorporated herein by reference in their entirety. As yet another alternative solid-phase microsequencing procedure, Nyren et al. (Analytical Biochemistry 208:171-175, 1993) described a method relying on the detection of DNA polymerase activity by an enzymatic luminometric inorganic pyrophosphate detection assay (ELIDA).
Pastinen et al. (Genome research 7:606-614, 1997), the disclosure of which is incorporated herein by reference in its entirety, describe a method for multiplex detection of single nucleotide polymorphism in which the solid phase minisequencing principle is applied to an oligonucleotide array format. High-density arrays of DNA probes attached to a solid support (DNA chips) are further described in IIIC.5.
In one aspect the present invention provides polynucleotides and methods to genotype one or more biallelic markers of the present invention by performing a microsequencing assay. Preferred microsequencing primers include those being featured Table 16. It will be appreciated that the microsequencing primers listed in Table 16 are merely exemplary and that, any primer having a 3xe2x80x2 end immediately adjacent to a polymorphic nucleotide may be used. Similarly, it will be appreciated that microsequencing analysis may be performed for any biallelic marker or any combination of biallelic markers of the present invention. One aspect of the present invention is a solid support which includes one or more microsequencing primers listed in Table 16, or fragments comprising at least 8, at least 12, at least 15, or at least 20 consecutive nucleotides thereof and having a 3xe2x80x2 terminus immediately upstream of the corresponding biallelic marker, for determining the identity of a nucleotide at biallelic marker site.
Mismatch Detection Assays Based on Polymerases and Ligases
In one aspect the present invention provides polynucleotides and methods to determine the allele of one or more biallelic markers of the present invention in a biological sample, by mismatch detection assays based on polymerases and/or ligases. These assays are based on the specificity of polymerases and ligases. Polymerization reactions places particularly stringent requirements on correct base pairing of the 3xe2x80x2 end of the amplification primer and the joining of two oligonucleotides hybridized to a target DNA sequence is quite sensitive to mismatches close to the ligation site, especially at the 3xe2x80x2 end. The terms xe2x80x9cenzyme based mismatch detection assayxe2x80x9d are used herein to refer to any method of determining the allele of a biallelic marker based on the specificity of ligases and polymerases. Preferred methods are described below. Methods, primers and various parameters to amplify DNA fragments comprising biallelic markers of the present invention are further described above in III.B.
Allele Specific Amplification
Discrimination between the two alleles of a biallelic marker can also be achieved by allele specific amplification, a selective strategy, whereby one of the alleles is amplified without amplification of the other allele. This is accomplished by placing a polymorphic base at the 3xe2x80x2 end of one of the amplification primers. Because the extension forms from the 3xe2x80x2 end of the primer, a mismatch at or near this position has an inhibitory effect on amplification. Therefore, under appropriate amplification conditions, these primers only direct amplification on their complementary allele. Designing the appropriate allele-specific primer and the corresponding assay conditions are well with the ordinary skill in the art.
Ligation Amplification Based Methods
The xe2x80x9cOligonucleotide Ligation Assayxe2x80x9d (OLA) uses two oligonucleotides which are designed to be capable of hybridizing to abutting sequences of a single strand of a target molecules. One of the oligonucleotides is biotinylated, and the other is detectably labeled. If the precise complementary sequence is found in a target molecule, the oligonucleotides will hybridize such that their termini abut, and create a ligation substrate that can be captured and detected. OLA is capable of detecting biallelic markers and may be advantageously combined with PCR as described by Nickerson D. A. et al. (Proc. Natl. Acad. Sci. U.S.A. 87:8923-8927, 1990)., the disclosure of which is incorporated herein by reference in its entirety in this method, PCR is used to achieve the exponential amplification of target DNA, which is then detected using OLA.
Other methods which are particularly suited for the detection of biallelic markers include LCR (ligase chain reaction), Gap LCR (GLCR) which are described above in III.B. As mentioned above LCR uses two pairs of probes to exponentially amplify a specific target. The sequences of each pair of oligonucleotides, is selected to permit the pair to hybridize to abutting sequences of the same strand of the target. Such hybridization forms a substrate for a template-dependant ligase. In accordance with the present invention, LCR can be performed with oligonucleotides having the proximal and distal sequences of the same strand of a biallelic marker site. In one embodiment, either oligonucleotide will be designed to include the biallelic marker site. In such an embodiment, the reaction conditions are selected such that the oligonucleotides can be ligated together only if the target molecule either contains or lacks the specific nucleotide(s) that is complementary to the biallelic marker on the oligonucleotide. In an alternative embodiment, the oligonucleotides will not include the biallelic marker, such that when they hybridize to the target molecule, a xe2x80x9cgapxe2x80x9d is created as described in WO 90/01069, the disclosure of which is incorporated herein by reference in its entirety. This gap is then xe2x80x9cfilledxe2x80x9d with complementary dNTPs (as mediated by DNA polymerase), or by an additional pair of oligonucleotides. Thus at the end of each cycle, each single strand has a complement capable of serving as a target during the next cycle and exponential allele-specific amplification of the desired sequence is obtained.
Ligase/Polymerase-mediated Genetic Bit Analysis(trademark) is another method for determining the identity of a nucleotide at a preselected site in a nucleic acid molecule (WO 95/21271), the disclosure of which is incorporated herein by reference in its entirety. This method involves the incorporation of a nucleoside triphosphate that is complementary to the nucleotide present at the preselected site onto the terminus of a primer molecule, and their subsequent ligation to a second oligonucleotide. The reaction is monitored by detecting a specific label attached to the reaction""s solid phase or by detection in solution.
Hybridization Assay Methods
A preferred method of determining the identity of the nucleotide present at a biallelic marker site involves nucleic acid hybridization. The hybridization probes, which can be conveniently used in such reactions, preferably include the probes defined herein. Any hybridization assay may be used including Southern hybridization, Northern hybridization, dot blot hybridization and solid-phase hybridization (see Sambrook et al., Molecular Cloningxe2x80x94A Laboratory Manual, Second Edition, Cold Spring Harbor Press, N.Y., 1989), the disclosure of which is incorporated herein by reference in its entirety.
Hybridization refers to the formation of a duplex structure by two single stranded nucleic acids due to complementary base pairing. Hybridization can occur between exactly complementary nucleic acid strands or between nucleic acid strands that contain minor regions of mismatch. Specific probes can be designed that hybridize to one form of a biallelic marker and not to the other and therefore are able to discriminate between different allelic forms. Allele-specific probes are often used in pairs, one member of a pair showing perfect match to a target sequence containing the original allele and the other showing a perfect match to the target sequence containing the alternative allele. Hybridization conditions should be sufficiently stringent that there is a significant difference in hybridization intensity between alleles, and preferably an essentially binary response, whereby a probe hybridizes to only one of the alleles. Stringent, sequence specific hybridization conditions, under which a probe will hybridize only to the exactly complementary target sequence are well known in the art (Sambrook et al., Molecular Cloningxe2x80x94A Laboratory Manual, Second Edition, Cold Spring Harbor Press, N.Y., 1989), the disclosures of which are incorporated herein by reference in their entirety. Stringent conditions are sequence dependent and will be different in different circumstances. Generally, stringent conditions are selected to be about 5xc2x0 C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. By way of example and not limitation, procedures using conditions of high stringency are as follows: Prehybridization of filters containing DNA is carried out for 8 h to overnight at 65xc2x0 C. in buffer composed of 6xc3x97SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA, and 500 xcexcg/ml denatured salmon sperm DNA. Filters are hybridized for 48 h at 65xc2x0 C., the preferred hybridization temperature, in prehybridization mixture containing 100 xcexcg/ml denatured salmon sperm DNA and 5-20xc3x97106 cpm of 32P-labeled probe. Alternatively, the hybridization step can be performed at 65xc2x0 C. in the presence of SSC buffer, 1xc3x97SSC corresponding to 0.15M NaCl and 0.05 M Na citrate. Subsequently, filter washes can be done at 37xc2x0 C. for 1 h in a solution containing 2xc3x97SSC, 0.01% PVP, 0.01% Ficoll, and 0.01% BSA, followed by a wash in 0.1xc3x97SSC at 50xc2x0 C. for 45 min. Alternatively, filter washes can be performed in a solution containing 2xc3x97SSC and 0.1% SDS, or 0.5xc3x97SSC and 0.1% SDS, or 0.1xc3x97SSC and 0.1% SDS at 68xc2x0 C. for 15 minute intervals. Following the wash steps, the hybridized probes are detectable by autoradiography. By way of example and not limitation, procedures using conditions of intermediate stringency are as follows: Filters containing DNA are prehybridized, and then hybridized at a temperature of 60xc2x0 C. in the presence of a 5xc3x97SSC buffer and labeled probe. Subsequently, filters washes are performed in a solution containing 2xc3x97SSC at 50xc2x0 C. and the hybridized probes are detectable by autoradiography. Other conditions of high and intermediate stringency which may be used are well known in the art and as cited in Sambrook et al. (Molecular Cloningxe2x80x94A Laboratory Manual, Second Edition, Cold Spring Harbor Press, N.Y., 1989) and Ausubel et al. (Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, N.Y., 1989).
Although such hybridizations can be performed in solution, it is preferred to employ a solid-phase hybridization assay. The target DNA comprising a biallelic marker of the present invention may be amplified prior to the hybridization reaction. The presence of a specific allele in the sample is determined by detecting the presence or the absence of stable hybrid duplexes formed between the probe and the target DNA. The detection of hybrid duplexes can be carried out by a number of methods. Various detection assay formats are well known which utilize detectable labels bound to either the target or the probe to enable detection of the hybrid duplexes. Typically, hybridization duplexes are separated from unhybridized nucleic acids and the labels bound to the duplexes are then detected. Those skilled in the art will recognize that wash steps may be employed to wash away excess target DNA or probe. Standard heterogeneous assay formats are suitable for detecting the hybrids using the labels present on the primers and probes.
Two recently developed assays allow hybridization-based allele discrimination with no need for separations or washes (see Landegren U. et al., Genome Research, 8:769-776,1998), the disclosure of which is incorporated herein by reference in its entirety. The TaqMan assay takes advantage of the 5xe2x80x2 nuclease activity of Taq DNA polymerase to digest a DNA probe annealed specifically to the accumulating amplification product. TaqMan probes are labeled with a donor-acceptor dye pair that interacts via fluorescence energy transfer. Cleavage of the TaqMan probe by the advancing polymerase during amplification dissociates the donor dye from the quenching acceptor dye, greatly increasing the donor fluorescence. All reagents necessary to detect two allelic variants can be assembled at the beginning of the reaction and the results are monitored in real time (see Livak et al., Nature Genetics, 9:341-342, 1995), the disclosure of which is incorporated herein by reference in its entirety. In an alternative homogeneous hybridization-based procedure, molecular beacons are used for allele discriminations. Molecular beacons are hairpin-shaped oligonucleotide probes that report the presence of specific nucleic acids in homogeneous solutions. When they bind to their targets they undergo a conformational reorganization that restores the fluorescence of an internally quenched fluorophore (Tyagi et al., Nature Biotechnology, 16:49-53, 1998)., the disclosure of which is incorporated herein by reference in its entirety
The polynucleotides provided herein can be used in hybridization assays for the detection of biallelic marker alleles in biological samples. These probes are characterized in that they preferably comprise between 8 and 50 nucleotides, and in that they are sufficiently complementary to a sequence comprising a biallelic marker of the present invention to hybridize thereto and preferably sufficiently specific to be able to discriminate the targeted sequence for only one nucleotide variation. The GC content in the probes of the invention usually ranges between 10 and 75%, preferably between 35 and 60%, and more preferably between 40 and 55%. The length of these probes can range from 10, 15, 20, or 30 to at least 100 nucleotides, preferably from 10 to 50, more preferably from 18 to 35 nucleotides. A particularly preferred probe is 25 nucleotides in length. Preferably the biallelic marker is within 4 nucleotides of the center of the polynucleotide probe. In particularly preferred probes the biallelic marker is at the center of said polynucleotide. Shorter probes may lack specificity for a target nucleic acid sequence and generally require cooler temperatures to form sufficiently stable hybrid complexes with the template. Longer probes are expensive to produce and can sometimes self-hybridize to form hairpin structures. Methods for the synthesis of oligonucleotide probes have been described above and can be applied to the probes of the present invention.
Preferably the probes of the present invention are labeled or immobilized on a solid support. Labels and solid supports are further described in I. Detection probes are generally nucleic acid sequences or uncharged nucleic acid analogs such as, for example peptide nucleic acids which are disclosed in International Patent Application WO 92/20702, d, morpholino analogs which are described in U.S. Pat. Nos. 5,185,444; 5,034,506 and 5,142,047, the disclosures of which are incorporated herein by reference in their entirety. The probe may have to be rendered xe2x80x9cnon-extendablexe2x80x9d in that additional dNTPs cannot be added to the probe. In and of themselves analogs usually are non-extendable and nucleic acid probes can be rendered non-extendable by modifying the 3xe2x80x2 end of the probe such that the hydroxyl group is no longer capable of participating in elongation. For example, the 3xe2x80x2 end of the probe can be functionalized with the capture or detection label to thereby consume or otherwise block the hydroxyl group. Alternatively, the 3xe2x80x2 hydroxyl group simply can be cleaved, replaced or modified, U.S. patent application Ser. No. 07/049,061 filed Apr. 19, 1993 describes modifications, which can be used to render a probe non-extendable.
The probes of the present invention are useful for a number of purposes. They can be used in Southern hybridization to genomic DNA or Northern hybridization to mRNA. The probes can also be used to detect PCR amplification products. By assaying the hybridization to an allele specific probe, one can detect the presence or absence of a biallelic marker allele in a given sample.
High-Throughput parallel hybridizations in array format are specifically encompassed within xe2x80x9chybridization assaysxe2x80x9d and are described below.
Hybridization to Addressable Arrays of Oligonucleotides
Hybridization assays based on oligonucleotide arrays rely on the differences in hybridization stability of short oligonucleotides to perfectly matched and mismatched target sequence variants. Efficient access to polymorphism information is obtained through a basic structure comprising high-density arrays of oligonucleotide probes attached to a solid support (the chip) at selected positions. Each DNA chip can contain thousands to millions of individual synthetic DNA probes arranged in a grid-ike pattern and miniaturized to the size of a dime.
The chip technology has already been applied with success in numerous cases. For example, the screening of mutations has been undertaken in the BRCA1 gene, in S. cerevisiae mutant strains, and in the protease gene of HIV-1 virus (Hacia et al., Nature Genetics, 14(4):441-447, 1996; Shoemaker et al., Nature Genetics, 14(4):450-456, 1996 ; Kozal et al., Nature Medicine, 2:753-759, 1996)the disclosures of which are incorporated herein by reference in their entirety. Chips of various formats for use in detecting biallelic polymorphisms can be produced on a customized basis by Affymetrix (GeneChip(trademark)), Hyseq (HyChip and HyGnostics), and Protogene Laboratories.
In general, these methods employ arrays of oligonucleotide probes that are complementary to target nucleic acid sequence segments from an individual which, target sequences include a polymorphic marker. EP785280, the disclosure of which is incorporated herein by reference in its entirety, describes a tiling strategy for the detection of single nucleotide polymorphisms. Briefly, arrays may generally be xe2x80x9ctiledxe2x80x9d for a large number of specific polymorphisms. By xe2x80x9ctilingxe2x80x9d is generally meant the synthesis of a defined set of oligonucleotide probes which is made up of a sequence complementary to the target sequence of interest, as well as preselected variations of that sequence, e.g., substitution of one or more given positions with one or more members of the basis set of monomers, i.e. nucleotides. Tiling strategies are further described in PCT application No. WO 95/11995, the disclosure of which is incorporated herein by reference in its entirety. In a particular aspect, arrays are tiled for a number of specific, identified biallelic marker sequences. In particular the array is tiled to include a number of detection blocks, each detection block being specific for a specific biallelic marker or a set of biallelic markers. For example, a detection block may be tiled to include a number of probes, which span the sequence segment that includes a specific polymorphism. To ensure probes that are complementary to each allele, the probes are synthesized in pairs differing at the biallelic marker. In addition to the probes differing at the polymorphic base, monosubstituted probes are also generally tiled within the detection block. These monosubstituted probes have bases at and up to a certain number of bases in either direction from the polymorphism, substituted with the remaining nucleotides (selected from A, T, G, C and U). Typically the probes in a tiled detection block will include substitutions of the sequence positions up to and including those that are 5 bases away from the biallelic marker. The monosubstituted probes provide internal controls for the tiled array, to distinguish actual hybridization from artefactual cross-hybridization. Upon completion of hybridization with the target sequence and washing of the array, the array is scanned to determine the position on the array to which the target sequence hybridizes. The hybridization data from the scanned array is then analyzed to identify which allele or alleles of the biallelic marker are present in the sample. Hybridization and scanning may be carried out as described in PCT application No. WO 92/10092 and WO 95/11995 and U.S. Pat. No. 5,424,186, the disclosures of which are incorporated herein by reference in their entirety.
Thus, in some embodiments, the chips may comprise an array of nucleic acid sequences of fragments of about 15 nucleotides in length. In further embodiments, the chip may comprise an array including at least one of the sequences selected from the group consisting of SEQ ID No. 1-38, 40-54, 56-463, 465-487, 490-493 and the sequences complementary thereto; preferably SEQ ID Nos. 485-487, 494-531, 533-547, 549-846, 848-956, 958-977 and the sequences complementary thereto; or a fragment thereof at least about 8 consecutive nucleotides, preferably 10, 15, 20, more preferably 25, 30, 40, 47, or 50 consecutive nucleotides. In some embodiments, the chip may comprise an array of at least 2, 3, 4, 5, 6, 7, 8 or more of these polynucleotides of the invention. Solid supports and polynucleotides of the present invention attached to solid supports are further described in I. xe2x80x9cBiallelic Markers and Polynucleotides Comprising Biallelic Markers.xe2x80x9d
Integrated Systems
Another technique, which may be used to analyze polymorphisms, includes multicomponent integrated systems, which miniaturize and compartmentalize processes such as PCR and capillary electrophoresis reactions in a single functional device. An example of such technique is disclosed in U.S. Pat. No. 5,589,136, the disclosure of which is incorporated herein by reference in its entirety, which describes the integration of PCR amplification and capillary electrophoresis in chips.
Integrated systems can be envisaged mainly when microfluidic systems are used. These systems comprise a pattern of microchannels designed onto a glass, silicon, quartz, or plastic wafer included on a microchip. The movements of the samples are controlled by electric, electroosmotic or hydrostatic forces applied across different areas of the microchip. For genotyping biallelic markers, the microfluidic system may integrate nucleic acid amplification, microsequencing, capillary electrophoresis and a detection method such as laser-induced fluorescence detection.
IV. Methods of Genetic Analysis Using the Biallelic Markers of the Present Invention
Different methods are available for the genetic analysis of complex traits (see Lander and Schork, Science, 265, 2037-2048, 1994), the disclosure of which is incorporated herein by reference in its entirety. The search for disease-susceptibility genes is conducted using two main methods: the linkage approach in which evidence is sought for cosegregation between a locus and a putative trait locus using family studies, and the association approach in which evidence is sought for a statistically significant association between an allele and a trait or a trait causing allele (Khoury J. et al., Fundamentals of Genetic Epidemiology, Oxford University Press, NY, 1993), the disclosure of which is incorporated herein by reference in its entirety. In general, the biallelic markers of the present invention find use in any method known in the art to demonstrate a statistically significant correlation between a genotype and a phenotype. The biallelic markers may be used in parametric and non-parametric linkage analysis methods. Preferably, the biallelic markers of the present invention are used to identify genes associated with detectable traits using association studies, an approach which does not require the use of affected families and which permits the identification of genes associated with complex and sporadic traits.
The genetic analysis using the biallelic markers of the present invention may be conducted on any scale. The whole set of biallelic markers of the present invention or any subset of biallelic markers of the present invention may be used. In some embodiments a subset of biallelic markers corresponding to one or several candidate genes of the present invention may be used. In other embodiments a subset of biallelic markers corresponding to candidate genes from a given metabolic pathway may be used. Such pathways include glucoronidation and glutathione conjugation. Alternatively, a subset of biallelic markers of the present invention localised on a specific chromosome segment may be used. Further, any set of genetic markers including a biallelic marker of the present invention may be used. A set of biallelic polymorphisms that, could be used as genetic markers in combination with the biallelic markers of the present invention, has been described in WO 98/20165, the disclosure of which is incorporated herein by reference in its entirety. As mentioned above, the biallelic markers of the present invention may be included in any complete or partial genetic map of the human genome. These different uses are specifically contemplated in the present invention and claims.
A. Linkage Analysis
Linkage analysis is based upon establishing a correlation between the transmission of genetic markers and that of a specific trait throughout generations within a family. Thus, the aim of linkage analysis is to detect marker loci that show cosegregation with a trait of interest in pedigrees.
Parametric Methods
When data are available from successive generations there is the opportunity to study the degree of linkage between pairs of loci. Estimates of the recombination fraction enable loci to be ordered and placed onto a genetic map. With loci that are genetic markers, a genetic map can be established, and then the strength of linkage between markers and traits can be calculated and used to indicate the relative positions of markers and genes affecting those traits (Weir, B. S., Genetic data Analysis II: Methods for Discrete population genetic Data, Sinauer Assoc., Inc., Sunderland, Mass., USA, 1996), the disclosure of which is incorporated herein by reference in its entirety. The classical method for linkage analysis is the logarithm of odds (lod) score method (see Morton N. E., Am.J. Hum. Genet., 7:277-318, 1955; Ott J., Analysis of Human Genetic Linkage, John Hopkins University Press, Baltimore, 1991), the disclosures of which are incorporated herein by reference in their entirety. Calculation of lod scores requires specification of the mode of inheritance for the disease (parametric method). Generally, the length of the candidate region identified using linkage analysis is between 2 and 20 Mb. Once a candidate region is identified as described above, analysis of recombinant individuals using additional markers allows further delineation of the candidate region. Linkage analysis studies have generally relied on the use of a maximum of 5,000 microsatellite markers, thus limiting the maximum theoretical attainable resolution of linkage analysis to about 600 kb on average.
Linkage analysis has been successfully applied to map simple genetic traits that show clear Mendelian inheritance patterns and have a high penetrance (i.e., the ratio between the number of trait positive carriers of allele a and the total number of a carriers in the population). However, parametric linkage analysis suffers from a variety of drawbacks. First, it is limited by its reliance on the choice of a genetic model suitable for each studied trait. Furthermore, as already mentioned, the resolution attainable using linkage analysis is limited, and complementary studies are required to refine the analysis of the typical 2 Mb to 20 Mb regions initially identified through linkage analysis. In addition, parametric linkage analysis approaches have proven difficult when applied to complex genetic traits, such as those due to the combined action of multiple genes and/or environmental factors. It is very difficult to model these factors adequately in a lod score analysis. In such cases, too large an effort and cost are needed to recruit the adequate number of affected families required for applying linkage analysis to these situations, as recently discussed by Risch, N. and Merikangas, K. (Science, 273:1516-1517, 1996), the disclosure of which is incorporated herein by reference in its entirety.
Non-parametric Methods
The advantage of the so-called non-parametric methods for linkage analysis is that they do not require specification of the mode of inheritance for the disease, they tend to be more useful for the analysis of complex traits. In non-parametric methods, one tries to prove that the inheritance pattern of a chromosomal region is not consistent with random Mendelian segregation by showing that affected relatives inherit identical copies of the region more often than expected by chance. Affected relatives should show excess xe2x80x9callele sharingxe2x80x9d even in the presence of incomplete penetrance and polygenic inheritance. In non-parametric linkage analysis the degree of agreement at a marker locus in two individuals can be measured either by the number of alleles identical by state (IBS) or by the number of alleles identical by descent (IBD). Affected sib pair analysis is a well-known special case and is the simplest form of these methods.
The biallelic markers of the present invention may be used in both parametric and non-parametric linkage analysis. Preferably biallelic markers may be used in non-parametric methods which allow the mapping of genes involved in complex traits. The biallelic markers of the present invention may be used in both IBD- and IBS-methods to map genes affecting a complex trait. In such studies, taking advantage of the high density of biallelic markers, several adjacent biallelic marker loci may be pooled to achieve the efficiency attained by multi-allelic markers (Zhao et al., Am. J. Hum. Genet., 63:225-240, 1998), the disclosure of which is incorporated herein by reference in its entirety.
However, both parametric and non-parametric linkage analysis methods analyse affected relatives, they tend to be of limited value in the genetic analysis of drug responses or in the analysis of side effects to treatments. This type of analysis is impractical in such cases due to the lack of availability of familial cases. In fact, the likelihood of having more than one individual in a family being exposed to the same drug at the same time is extremely low.
B. Population Association Studies
The present invention comprises methods for identifying one or several genes among a set of candidate genes that are associated with a detectable trait using the biallelic markers of the present invention. In one embodiment the present invention comprises methods to detect an association between a biallelic marker allele or a biallelic marker haplotype and a trait. Further, the invention comprises methods to identify a trait causing allele in linkage disequilibrium with any biallelic marker allele of the present invention.
As described above, alternative approaches can be employed to perform association studies: genome-wide association studies, candidate region association studies and candidate gene association studies. In a preferred embodiment, the biallelic markers of the present invention are used to perform candidate gene association studies. The candidate gene analysis clearly provides a short-cut approach to the identification of genes and gene polymorphisms related to a particular trait when some information concerning the biology of the trait is available. Further, the biallelic markers of the present invention may be incorporated in any map of genetic markers of the human genome in order to perform genome-wide association studies. Methods to generate a high-density map of biallelic markers has been described in U.S. Provisional Patent application serial No. 60/082,614. The biallelic markers of the present invention may further be incorporated in any map of a specific candidate region of the genome (a specific chromosome or a specific chromosomal segment for example).
As mentioned above, association studies may be conducted within the general population and are not limited to studies performed on related individuals in affected families. Association studies are extremely valuable as they permit the analysis of sporadic or multifactor traits. Moreover, association studies represent a powerful method for fine-scale mapping enabling much finer mapping of trait causing alleles than linkage studies. Studies based on pedigrees often only narrow the location of the trait causing allele. Association studies using the biallelic markers of the present invention can therefore be used to refine the location of a trait causing allele in a candidate region identified by Linkage Analysis methods. Moreover, once a chromosome segment of interest has been identified, the presence of a candidate gene such as a candidate gene of the present invention, in the region of interest can provide a shortcut to the identification of the trait causing allele. Biallelic markers of the present invention can be used to demonstrate that a candidate gene is associated with a trait. Such uses are specifically contemplated in the present invention and claims.
Determining the Frequency of a Biallelic Marker Allele or of a Biallelic Marker Haplotype in a Population
Association studies explore the relationships among frequencies for sets of alleles between loci.
Determining the Frequency of an Allele in a Population
Allelic frequencies of the biallelic markers in a population can be determined using one of the methods described above under the heading xe2x80x9cMethods for genotyping an individual for biallelic markersxe2x80x9d, or any genotyping procedure suitable for this intended purpose. Genotyping pooled samples or individual samples can determine the frequency of a biallelic marker allele in a population. One way to reduce the number of genotypings required is to use pooled samples. A major obstacle in using pooled samples is in terms of accuracy and reproducibility for determining accurate DNA concentrations in setting up the pools. Genotyping individual samples provides higher sensitivity, reproducibility and accuracy and; is the preferred method used in the present invention. Preferably, each individual is genotyped separately and simple gene counting is applied to determine the frequency of an allele of a biallelic marker or of a genotype in a given population.
Determining the Frequency of a Haplotype in a Population
The gametic phase of haplotypes is unknown when diploid individuals are heterozygous at more than one locus. Using genealogical information in families gametic phase can sometimes be inferred (Perlin et al., Am. J. Hum. Genet., 55:777-787, 1994), the disclosure of which is incorporated herein by reference in its entirety. When no genealogical information is available different strategies may be used. One possibility is that the multiple-site heterozygous diploids can be eliminated from the analysis, keeping only the homozygotes and the single-site heterozygote individuals, but this approach might lead to a possible bias in the sample composition and the underestimation of low-frequency haplotypes. Another possibility is that single chromosomes can be studied independently, for example, by asymmetric PCR amplification (see Newton et al., Nucleic Acids Res., 17:2503-2516, 1989; Wu et al., Proc. Natl. Acad. Sci. USA, 86:2757, 1989), the disclosures of which are incorporated herein by reference in their entirety, or by isolation of single chromosome by limit dilution followed by PCR amplification (see Ruano et al., Proc. Natl. Acad. Sci. USA, 87:6296-6300, 1990), the disclosure of which is incorporated herein by reference in its entirety. Further, a sample may be haplotyped for sufficiently close biallelic markers by double PCR amplification of specific alleles (Sarkar, G. and Sommer S. S., Biotechniques, 1991), the disclosure of which is incorporated herein by reference in its entirety. These approaches are not entirely satisfying either because of their technical complexity, the additional cost they entail, their lack of generalisation at a large scale, or the possible biases they introduce. To overcome these difficulties, an algorithm to infer the phase of PCR-amplified DNA genotypes introduced by Clark A. G. (Mol. Biol. Evol., 7:111-122, 1990), the disclosure of which is incorporated herein by reference in its entirety, may be used. Briefly, the principle is to start filling a preliminary list of haplotypes present in the sample by examining unambiguous individuals, that is, the complete homozygotes and the single-site heterozygotes. Then other individuals in the same sample are screened for the possible occurrence of previously recognised haplotypes. For each positive identification, the complementary haplotype is added to the list of recognised haplotypes, until the phase information for all individuals is either resolved or identified as unresolved. This method assigns a single haplotype to each multiheterozygous individual, whereas several haplotypes are possible when there are more than one heterozygous site. Alternatively, one can use methods estimating haplotype frequencies in a population without assigning haplotypes to each individual. Preferably, a method based on an expectation-maximization (EM) algorithm (Dempster et al., J. R. Stat. Soc., 39B: 1-38, 1977), the disclosure of which is incorporated herein by reference in its entirety, leading to maximum-likelihood estimates of haplotype frequencies under the assumption of Hardy-Weinberg proportions (random mating) is used (see Excoffier L. and Slatkin M., Mol. Biol. Evol., 12(5): 921-927, 1995), the disclosure of which is incorporated herein by reference in its entirety. The EM algorithm is a generalised iterative maximum-likelihood approach to estimation that is useful when data are ambiguous and/or incomplete. The EM algorithm is used to resolve heterozygotes into haplotypes. Haplotype estimations are further described below under the heading xe2x80x9cStatistical methods.xe2x80x9d Any other method known in the art to determine or to estimate the frequency of a haplotype in a population may also be used.
Linkage Disequilibrium Analysis
Linkage disequilibrium is the non-random association of alleles at two or more loci and represents a powerful tool for mapping genes involved in disease traits (see Ajioka R. S. et al., Am. J. Hum. Genet., 60:1439-1447, 1997), the disclosure of which is incorporated herein by reference in its entirety. Biallelic markers, because they are densely spaced in the human genome and can be genotyped in more numerous numbers than other types of genetic markers (such as RFLP or VNTR markers), are particularly useful in genetic analysis based on linkage disequilibrium. The biallelic markers of the present invention may be used in any linkage disequilibrium analysis method known in the art.
Briefly, when a disease mutation is first introduced into a population (by a new mutation or the immigration of a mutation carrier), it necessarily resides on a single chromosome and thus on a single xe2x80x9cbackgroundxe2x80x9d or xe2x80x9cancestralxe2x80x9d haplotype of linked markers. Consequently, there is complete disequilibrium between these markers and the disease mutation: one finds the disease mutation only in the presence of a specific set of marker alleles. Through subsequent generations recombinations occur between the disease mutation and these marker polymorphisms, and the disequilibrium gradually dissipates. The pace of this dissipation is a function of the recombination frequency, so the markers closest to the disease gene will manifest higher levels of disequilibrium than those that are further away. When not broken up by recombination, xe2x80x9cancestralxe2x80x9d haplotypes and linkage disequilibrium between marker alleles at different loci can be tracked not only through pedigrees but also through populations. Linkage disequilibrium is usually seen as an association between one specific allele at one locus and another specific allele at a second locus.
The pattern or curve of disequilibrium between disease and marker loci is expected to exhibit a maximum that occurs at the disease locus. Consequently, the amount of linkage disequilibrium between a disease allele and closely linked genetic markers may yield valuable information regarding the location of the disease gene. For fine-scale mapping of a disease locus, it is useful to have some knowledge of the patterns of linkage disequilibrium that exist between markers in the studied region. As mentioned above the mapping resolution achieved through the analysis of linkage disequilibrium is much higher than that of linkage studies. The high density of biallelic markers combined with linkage disequilibrium analysis provides powerful tools for fine-scale mapping. Different methods to calculate linkage disequilibrium are described below under the heading xe2x80x9cStatistical Methods.xe2x80x9d
Population-Based Case-Control Studies of Trait-Marker Associations
As mentioned above, the occurrence of pairs of specific alleles at different loci on the same chromosome is not random and the deviation from random is called linkage disequilibrium. Association studies focus on population frequencies and rely on the phenomenon of linkage disequilibrium. If a specific allele in a given gene is directly involved in causing a particular trait, its frequency will be statistically increased in an affected (trait positive) population, when compared to the frequency in a trait negative population or in a random control population. As a consequence of the existence of linkage disequilibrium, the frequency of all other alleles present in the haplotype carrying the trait-causing allele will also be increased in trait positive individuals compared to trait negative individuals or random controls. Therefore, association between the trait and any allele (specifically a biallelic marker allele) in linkage disequilibrium with the trait-causing allele will suffice to suggest the presence of a trait-related gene in that particular region. Case-control populations can be genotyped for biallelic markers to identify associations that narrowly locate a trait causing allele. As any marker in linkage disequilibrium with one given marker associated with a trait will be associated with the trait. Linkage disequilibrium allows the relative frequencies in case-control populations of a limited number of genetic polymorphisms (specifically biallelic markers) to be analysed as an alternative to screening all possible functional polymorphisms in order to find trait-causing alleles. Association studies compare the frequency of marker alleles in unrelated case-control populations, and represent powerful tools for the dissection of complex traits.
Case-Control Populations (Inclusion Criteria)
Population-based association studies do not concern familial inheritance but compare the prevalence of a particular genetic marker, or a set of markers, in case-control populations. They are case-control studies based on comparison of unrelated case (affected or trait positive) individuals and unrelated control (unaffected or trait negative or random) individuals. Preferably the control group is composed of unaffected or trait negative individuals. Further, the control group is ethnically matched to the case population. Moreover, the control group is preferably matched to the case-population for the main known confusion factor for the trait under study (for example age-matched for an age-dependent trait). Ideally, individuals in the two samples are paired in such a way that they are expected to differ only in their disease status. In the following xe2x80x9ctrait positive populationxe2x80x9d, xe2x80x9ccase populationxe2x80x9d and xe2x80x9caffected populationxe2x80x9d are used interchangeably.
An important step in the dissection of complex traits using association studies is the choice of case-control populations (see Lander and Schork Science, 265, 2037-2048, 1994), the disclosure of which is incorporated herein by reference in its entirety. A major step in the choice of case-control populations is the clinical definition of a given trait or phenotype. Any genetic trait may be analysed by the association method proposed here by carefully selecting the individuals to be included in the trait positive and trait negative phenotypic groups. Four criteria are often useful: clinical phenotype, age at onset, family history and severity. The selection procedure for continuous or quantitative traits (such as blood pressure for example) involves selecting individuals at opposite ends of the phenotype distribution of the trait under study, so as to include in these trait positive and trait negative populations individuals with non-overlapping phenotypes. Preferably, case-control populations consist of phenotypically homogeneous populations. Trait positive and trait negative populations consist of phenotypically uniform populations of individuals representing each between 1 and 98%, preferably between 1 and 80%, more preferably between 1 and 50%, and more preferably between 1 and 30%, most preferably between 1 and 20% of the total population under study, and selected among individuals exhibiting non-overlapping phenotypes. The clearer the difference between the two trait phenotypes, the greater the probability of detecting an association with biallelic markers. The selection of those drastically different but relatively uniform phenotypes enables efficient comparisons in association studies and the possible detection of marked differences at the genetic level, provided that the sample sizes of the populations under study are significant enough.
In preferred embodiments, a first group of between 50 and 300 trait positive individuals, preferably about 100 individuals, are recruited according to their phenotypes. A similar number of trait negative individuals are included in such studies.
In the present invention, typical examples of inclusion criteria include a disease involving the metabolic conversion of xenobiotics or the evaluation of the response to a drug or side effects to treatment with drugs.
Suitable examples of association studies using biallelic markers including the biallelic markers of the present invention, are studies involving the following populations:
a case population suffering from a disease involving the metabolic conversion of xenobiotics and a healthy unaffected control population, or
a case population treated with therapeutic agents suffering from side-effects resulting from the treatment and a control population treated with the same agents showing no side-effects, or
a case population treated with therapeutic agents showing a beneficial response and a control population treated with same agents showing no beneficial response.
In a preferred embodiment, the trait considered was a side-effect upon drug treatment, the study involved two populations derived from a clinical study of the anti-asthmatic drug zileuton. The case population was composed of asthmatic individuals treated with zileuton showing zileuton-associated hepatotoxicity monitored by the serum level of alanine aminotransferase (ALT) and the control population was composed of asthmatic individuals treated with zileuton and having no increased serum level of ALT. Inclusion criteria and association between the biallelic markers of the present invention and zileuton-associated hepatotoxicity are further described below and in Example 4.
Association Analysis
The general strategy to perform association studies using biallelic markers derived from a region carrying a candidate gene is to scan two groups of individuals (case-control populations) in order to measure and statistically compare the allele frequencies of the biallelic markers of the present invention in both groups.
If a statistically significant association with a trait is identified for at least one or more of the analysed biallelic markers, one can assume that: either the associated allele is directly responsible for causing the trait (the associated allele is the trait causing allele), or more likely the associated allele is in linkage disequilibrium with the trait causing allele. The specific characteristics of the associated allele with respect to the candidate gene function usually gives further insight into the relationship between the associated allele and the trait (causal or in linkage disequilibrium). If the evidence indicates that the associated allele within the candidate gene is most probably not the trait causing allele but is in linkage disequilibrium with the real trait causing allele, then the trait causing allele can be found by sequencing the vicinity of the associated marker.
Association studies are usually run in two successive steps. In a first phase, the frequencies of a reduced number of biallelic markers from one or several candidate genes are determined in the trait positive and trait negative populations. hi a second phase of the analysis, the identity of the candidate gene and the position of the genetic loci responsible for the given trait is further refined using a higher density of markers from the relevant region. However, if the candidate gene under study is relatively small in length, as it is the case for many of the candidate genes analysed included in the present invention, a single phase may be sufficient to establish significant associations.
Haplotype Analysis
As described above, when a chromosome carrying a disease allele first appears in a population as a result of either mutation or migration, the mutant allele necessarily resides on a chromosome having a set of linked markers: the ancestral haplotype. This haplotype can be tracked through populations and its statistical association with a given trait can be analysed. Complementing single point (allelic) association studies with multi-point association studies also called haplotype studies increases the statistical power of association studies. Thus, a haplotype association study allows one to define the frequency and the type of the ancestral carrier haplotype. A haplotype analysis is important in that it increases the statistical power of an analysis involving individual markers.
In a first stage of a haplotype frequency analysis, the frequency of the possible haplotypes based on various combinations of the identified biallelic markers of the invention is determined. The haplotype frequency is then compared for distinct populations of trait positive and control individuals. The number of trait positive individuals, which should be, subjected to this analysis to obtain statistically significant results usually ranges between 30 and 300, with a preferred number of individuals ranging between 50 and 150. The same considerations apply to the number of unaffected individuals (or random control) used in the study. The results of this first analysis provide haplotype frequencies in case-control populations, for each evaluated haplotype frequency a p-value and an odd ratio are calculated. If a statistically significant association is found, the relative risk for an individual carrying the given haplotype of being affected with the trait under study can be approximated.
The preferred 2, 3 and 4 marker haplotypes of the invention are listed in Table 4 below:
The most preferred 2, 3 and 4 marker haplotypes of the invention are listed in Table 5 below:
Interaction Analysis
The biallelic markers of the present invention may also be used to identify patterns of biallelic markers associated with detectable traits resulting from polygenic interactions. The analysis of genetic interaction between alleles at unlinked loci requires individual genotyping using the techniques described herein. The analysis of allelic interaction among a selected set of biallelic markers with appropriate level of statistical significance can be considered as a haplotype analysis. Interaction analysis consists in stratifying the case-control populations with respect to a given haplotype for the first loci and performing a haplotype analysis with the second loci with each subpopulation.
Statistical methods used in association studies are further described below in xe2x80x9cStatistical Methods.xe2x80x9d
Testing for Linkage in the Presence of Association
The biallelic markers of the present invention may further be used in TDT (transmission/disequilibrium test). TDT tests for both linkage and association and is not affected by population stratification. TDT requires data for affected individuals and their parents or data from unaffected sibs instead of from parents (see Spielmann S. et al., Am. J. Hum. Genet., 52:506-516, 1993; Schaid D. J. et al., Genet. Epidemiol.,13:423-450, 1996, Spielmann S. and Ewens W. J., Am. J. Hum. Genet., 62:450-458, 1998), the disclosures of which are incorporated herein by reference in their entirety. Such combined tests generally reduce the false-positive errors produced by separate analyses.
C. Statistical Methods
In general, any method known in the art to test whether a trait and a genotype show a statistically significant correlation may be used.
Methods in Linkage Analysis
Statistical methods and computer programs useful for linkage analysis are well known to those skilled in the art (see Terwilliger J. D. and Ott J., Handbook of Human Genetic Linkage, John Hopkins University Press, London, 1994; Ott J., Analysis of Human Genetic Linkage, John Hopkins University Press, Baltimore, 1991), the disclosures of which are incorporated herein by reference in their entirety.
Methods to Estimate Haplotype Frequencies in a Population
As described above, when genotypes are scored, it is often not possible to distinguish heterozygotes so that haplotype frequencies cannot be easily inferred. When the gametic phase is not known, haplotype frequencies can be estimated from the multilocus genotypic data. Any method known to person skilled in the art can be used to estimate haplotype frequencies (see Lange K., Mathematical and Statistical Methods for Genetic Analysis, Springer, N.Y., 1997; Weir, B. S., Genetic data Analysis II: Methods for Discrete population genetic Data, Sinauer Assoc., Inc., Sunderland, Mass., USA, 1996), the disclosures of which are incorporated herein by reference in their entirety. Preferably, maximum-likelihood haplotype frequencies are computed using an Expectation-Maximization (EM) algorithm (see Dempster et al., J. R. Stat. Soc., 39B: 1-38, 1977; Excoffier L. and Slatkin M., Mol. Biol. Evol., 12(5): 921-927, 1995)., the disclosures of which are incorporated herein by reference in their entirety. This procedure is an iterative process aiming at obtaining maximum-likelihood estimates of haplotype frequencies from multi-locus genotype data when the gametic phase is unknown. Haplotype estimations are usually performed by applying the EM algorithm using, for example, the EM-HAPLO program (Hawley M. E. et al., Am. J. Phys. Anthropol., 18:104,1994) or the Arlequin program (Schneider et al., Arlequin: a software for population genetics data analysis, University of Geneva, 1997), the disclosure of which is incorporated herein by reference in its entirety. The EM algorithm is a generalised iterative maximum likelihood approach to estimation and is briefly described below.
In what follows, phenotypes will refer to multi-locus genotypes with unknown haplotypic phase. Genotypes will refer to mutli-locus genotypes with known haplotypic phase.
Suppose one has a sample of N unrelated individuals typed for K markers. The data observed are the unknown-phase K-locus phenotypes that can be categorized with F different phenotypes. Further, suppose that we have H possible haplotypes (in the case of K biallelic markers, we have for the maximum number of possible haplotypes H=2K). For phenotype j with cj possible genotypes, we have:                               P          j                =                                            ∑                              i                =                1                                            c                j                                      ⁢                          xe2x80x83                        ⁢                          P              ⁡                              (                                  genotype                  ⁡                                      (                    i                    )                                                  )                                              =                                    ∑                              i                =                1                                            c                j                                      ⁢                          xe2x80x83                        ⁢                                          P                ⁡                                  (                                                            h                      k                                        ,                                          h                      l                                                        )                                            .                                                          Equation        ⁢                  xe2x80x83                ⁢        1            
Here, Pj is the probability of the jth phenotype, and P(hk,hl) is the probability of the ith genotype composed of haplotypes hk and hl. Under random mating (i.e. Hardy-Weinberg Equilibrium), P(hkhl) is expressed as:
P(hk,hl)=P(hk)2 for hk=hl,
and
P(hk,hl)=2P(hk)P(hl) for hkxe2x89xa0hl.xe2x80x83xe2x80x83Equation 2
The E-M algorithm is composed of the following steps: First, the genotype frequencies are estimated from a set of initial values of haplotype frequencies. These haplotype frequencies are denoted P1(0), P2(0), P3(0), . . . , PH(0). The initial values for the haplotype frequencies may be obtained from a random number generator or in some other way well known in the art. This step is referred to the Expectation step. The next step in the method, called the Maximization step, consists of using the estimates for the genotype frequencies to re-calculate the haplotype frequencies. The first iteration haplotype frequency estimates are denoted by P1(1), P2(1), P3(1), . . . , PH(1). In general, the Expectation step at the sth iteration consists of calculating the probability of placing each phenotype into the different possible genotypes based on the haplotype frequencies of the previous iteration:                                                         P              ⁡                              (                                                      h                    k                                    ,                                      h                    l                                                  )                                                    (              s              )                                =                                                    n                j                            N                        ⁡                          [                                                                                          P                      j                                        ⁡                                          (                                                                        h                          k                                                ,                                                  h                          l                                                                    )                                                                            (                    s                    )                                                                    P                  j                                            ]                                      ,                            Equation        ⁢                  xe2x80x83                ⁢        3            
where nj is the number of individuals with the jth phenotype and Pj(hk,hl)(s) is the probability of genotype hk,hl in phenotype j. In the Maximization step, which is equivalent to the gene-counting method (Smith, Ann. Hum. Genet., 21:254-276, 1957), the haplotype frequencies are re-estimated based on the genotype estimates:                               P          t                      (                          s              +              1                        )                          =                              1            2                    ⁢                                    ∑                              j                =                1                            F                        ⁢                          xe2x80x83                        ⁢                                          ∑                                  i                  =                  1                                                  c                  j                                            ⁢                              xe2x80x83                            ⁢                                                δ                  it                                ⁢                                                                                                    P                        j                                            ⁡                                              (                                                                              h                            k                                                    ,                                                      h                            l                                                                          )                                                                                    (                      s                      )                                                        .                                                                                        Equation        ⁢                  xe2x80x83                ⁢        4            
Here, xcex4it is an indicator variable which counts the number of occurrences that haplotype t is present in ith genotype; it takes on values 0, 1, and 2.
The E-M iterations cease when the following criterion has been reached. Using Maximum Likelihood Estimation (MLE) theory, one assumes that the phenotypes j are distributed multinomially. At each iteration s, one can compute the likelihood function L. Convergence is achieved when the difference of the log-likehood between two consecutive iterations is less than some small number, preferably 10xe2x88x927.
Methods to Calculate Linkage Disequilibrium Between Markers
A number of methods can be used to calculate linkage disequilibrium between any two genetic positions, in practice linkage disequilibrium is measured by applying a statistical association test to haplotype data taken from a population.
Linkage disequilibrium between any pair of biallelic markers comprising at least one of the biallelic markers of the present invention (Mi, Mj) having alleles (ai/bi) at marker Mi and alleles (aj/bj) at marker Mj can be calculated for every allele combination (ai,aj; ai,bj; bi,aj and bi,bj), according to the Piazza formula:
xcex94aiaj=xcex84xe2x88x92(xcex84+xcex83)(xcex84+xcex82),
where:
xcex84=xe2x88x92 xe2x88x92=frequency of genotypes not having allele ai at Mi and not having allele aj at Mj 
xcex83=xe2x88x92+=frequency of genotypes not having allele ai at Mi and having allele aj at Mj 
xcex82=+xe2x88x92=frequency of genotypes having allele a1 at Mi and not having allele aj at Mj 
Linkage disequilibrium (LD) between pairs of biallelic markers (Mi, Mj) can also be calculated for every allele combination (ai,aj; ai,bj; bi,aj and bi,bj), according to the maximum-likelihood estimate (MLE) for delta (the composite genotypic disequilibrium coefficient), as described by Weir (Weir B. S., Genetic Data Analysis, Sinauer Ass. Eds, 1996). The MLE for the composite linkage disequilibrium is:
Daiaj=(2n1+n2+n3+n4/2)/Nxe2x88x922(pr(ai).pr(aj))
Where n1=xcexa3 phenotype (ai/ai, aj/aj), n2=xcexa3 phenotype (ai/ai, aj/bj), n3=xcexa3 phenotype (ai/bi, aj/aj), n4=xcexa3 phenotype (ai/bi, aj/bj) and N is the number of individuals in the sample.
This formula allows linkage disequilibrium between alleles to be estimated when only genotype, and not haplotype, data are available.
Another means of calculating the linkage disequilibrium between markers is as follows. For a couple of biallelic markers, Mi(ai/bi) and Mj(aj/bj), fitting the Hardy-Weinberg equilibrium, one can estimate the four possible haplotype frequencies in a given population according to the approach described above.
The estimation of gametic disequilibrium between ai and aj is simply:
Daiaj=pr(haplotype(ai,aj))xe2x88x92pr(ai).pr(aj).
Where pr(ai) is the probability of allele ai and pr(aj) is the probability of allele aj and where pr(haplotype (ai, aj)) is estimated as in Equation 3 above.
For a couple of biallelic marker only one measure of disequilibrium is necessary to describe the association between Mi and Mj.
Then a normalised value of the above is calculated as follows:
Dxe2x80x2aiaj=Daiaj/max(xe2x88x92pr(ai).pr(aj), xe2x88x92pr(bi).pr(bj)) with Daiaj less than 0
Dxe2x80x2aiaj=Daiaj/max(pr(bi).pr(aj), pr(ai).pr(bj)) with Daiaj greater than 0
The skilled person will readily appreciate that other LD calculation methods can be used without undue experimentation.
Linkage disequilibrium among a set of biallelic markers having an adequate heterozygosity rate can be determined by genotyping between 50 and 1000 unrelated individuals, preferably between 75 and 200, more preferably around 100.
Testing for Association
Methods for determining the statistical significance of a correlation between a phenotype and a genotype, in this case an allele at a biallelic marker or a haplotype made up of such alleles, may be determined by any statistical test known in the art and with any accepted threshold of statistical significance being required. The application of particular methods and thresholds of significance are well with in the skill of the ordinary practitioner of the art.
Testing for association is performed by determining the frequency of a biallelic marker allele in case and control populations and comparing these frequencies with a statistical test to determine if their is a statistically significant difference in frequency which would indicate a correlation between the trait and the biallelic marker allele under study. Similarly, a haplotype analysis is performed by estimating the frequencies of all possible haplotypes for a given set of biallelic markers in case and control populations, and comparing these frequencies with a statistical test to determine if their is a statistically significant correlation between the haplotype and the phenotype (trait) under study. Any statistical tool useful to test for a statistically significant association between a genotype and a phenotype may be used. Preferably the statistical test employed is a chi-square test with one degree of freedom. A P-value is calculated (the P-value is the probability that a statistic as large or larger than the observed one would occur by chance).
Statistical Significance
In preferred embodiments, significance for diagnosis purposes, either as a positive basis for further diagnostic tests or as a preliminary starting point for early preventive therapy, the p value related to a biallelic marker association is preferably about 1xc3x9710xe2x88x922 or less, more preferably about 1xc3x9710xe2x88x924 or less, for a single biallelic marker analysis and about 1xc3x9710xe2x88x923 or less, still more preferably 1xc3x9710xe2x88x926 or less and most preferably of about 1xc3x9710xe2x88x928 or less, for a haplotype analysis involving several markers. These values are believed to be applicable to any association studies involving single or multiple marker combinations.
The skilled person can use the range of values set forth above as a starting point in order to carry out association studies with biallelic markers of the present invention. In doing so, significant associations between the biallelic markers of the present invention and responses to drugs or side effects upon treatment with drugs or diseases involving the metabolic conversion of xenobiotics can be revealed and used for diagnosis and drug screening purposes.
Phenotypc Permutation
In order to confirm the statistical significance of the first stage haplotype analysis described above, it might be suitable to perform further analyses in which genotyping data from case-control individuals are pooled and randomised with respect to the trait phenotype. Each individual genotyping data is randomly allocated to two groups, which contain the same number of individuals as the case-control populations used to compile the data obtained in the first stage. A second stage haplotype analysis is preferably run on these artificial groups, preferably for the markers included in the haplotype of the first stage analysis showing the highest relative risk coefficient. This experiment is reiterated preferably at least between 100 and 10000 times. The repeated iterations allow the determination of the percentage of obtained haplotypes with a significant p-value level.
Assessment of Statistical Association
To address the problem of false positives similar analysis may be performed with the same case-control populations in random genomic regions. Results in random regions and the candidate region are compared as described in U.S. Provisional Patent Application entitled xe2x80x9cMethods, software and apparati for identifying genomic regions harbouring a gene associated with a detectable traitxe2x80x9d.
Evaluation of Risk Factors
The association between a risk factor (in genetic epidemiology the risk factor is the presence or the absence of a certain allele or haplotype at marker loci) and a disease is measured by the odds ratio (OR) and by the relative risk (RR). If P(R+) is the probability of developing the disease for individuals with R and P(Rxe2x88x92) is the probability for individuals without the risk factor, then the relative risk is simply the ratio of the two probabilities, that is:
RR=P(R+)/P(Rxe2x88x92)
In case-control studies, direct measures of the relative risk cannot be obtained because of the sampling design. However, the odds ratio allows a good approximation of the relative risk for low-incidence diseases and can be calculated:   OR  =            [                        F          +                          1          -                      F            +                              ]        /          [                        F          -                          (                      1            -                          F              -                                )                    ]      
F+ is the frequency of the exposure to the risk factor in cases and F- is the frequency of the exposure to the risk factor in controls. F+ and Fxe2x88x92 are calculated using the allelic or haplotype frequencies of the study and further depend on the underlying genetic model (dominant, recessive, additive . . . ).
One can further estimate the attributable risk (AR) which describes the proportion of individuals in a population exhibiting a trait due to a given risk factor. This measure is important in quantitating the role of a specific factor in disease etiology and in terms of the public health impact of a risk factor. The public health relevance of this measure lies in estimating the proportion of cases of disease in the population that could be prevented if the exposure of interest were absent. AR is determined as follows:
AR=PE(RRxe2x88x921)/(PE(RRxe2x88x921)+1)
AR is the risk attributable to a biallelic marker allele or a biallelic marker haplotype. PE is the frequency of exposure to an allele or a haplotype within the population at large; and RR is the relative risk which, is approximated with the odds ratio when the trait under study has a relatively low incidence in the general population.
D. Identification of Biallelic Markers in Linkage Disequilibrium with the Biallelic Markers of the Invention
Once a first biallelic marker has been identified in a genomic region of interest, the practitioner of ordinary skill in the art, using the teachings of the present invention, can easily identify additional biallelic markers in linkage disequilibrium with this first marker. As mentioned before any marker in linkage disequilibrium with a first marker associated with a trait will be associated with the trait. Therefore, once an association has been demonstrated between a given biallelic marker and a trait, the discovery of additional biallelic markers associated with this trait is of great interest in order to increase the density of biallelic markers in this particular region. The causal gene or mutation will be found in the vicinity of the marker or set of markers showing the highest correlation with the trait.
Identification of additional markers in linkage disequilibrium with a given marker involves: (a) amplifying a genomic fragment comprising a first biallelic marker from a plurality of individuals; (b) identifying of second biallelic markers in the genomic region harboring said first biallelic marker; (c) conducting a linkage disequilibrium analysis between said first biallelic marker and second biallelic markers; and (d) selecting said second biallelic markers as being in linkage disequilibrium with said first marker. Subcombinations comprising steps (b) and (c) are also contemplated.
Methods to identify biallelic markers and to conduct linkage disequilibrium analysis are described herein and can be carried out by the skilled person without undue experimentation. The present invention then also concerns biallelic markers which are in linkage disequilibrium with the specific biallelic markers shown in Table 11(A-B) and which are expected to present similar characteristics in terms of their respective association with a given trait.
E. Identification of Functional Mutations
Once a positive association is confirmed with a biallelic marker of the present invention, the associated candidate gene can be scanned for mutations by comparing the sequences of a selected number of trait positive and trait negative or control individuals. In a preferred embodiment, functional regions such as exons and splice sites, promoters and other regulatory regions of the candidate gene are scanned for mutations. Preferably, trait positive individuals carry the haplotype shown to be associated with the trait and trait negative individuals do not carry the haplotype or allele associated with the trait. The mutation detection procedure is essentially similar to that used for biallelic site identification.
The method used to detect such mutations generally comprises the following steps: (a) amplification of a region of the candidate gene comprising a biallelic marker or a group of biallelic markers associated with the trait from DNA samples of trait positive patients and trait negative controls; (b) sequencing of the amplified region; (c) comparison of DNA sequences from trait-positive patients and trait-negative controls; and (d) determination of mutations specific to trait-positive patients. Subcombinations which comprise steps (b) and (c) are specifically contemplated.
It is preferred that candidate polymorphisms be then verified by screening a larger population of cases and controls by means of any genotyping procedure such as those described herein, preferably using a microsequencing technique in an individual test format. Polymorphisms are considered as candidate mutations when present in cases and controls at frequencies compatible with the expected association results.
Identification of mutations and low frequency polymorphisms in exons 3-5, in the 5xe2x80x2UTR region and in the 3xe2x80x2 flanking region of the MGST-II gene is further described in Example 5. Eight polymorphisms were identified in the region of the MGST-II gene that was scanned. Three mutations were identified in the 3xe2x80x2UTR region. One mutation in exon 4 causes an amino acid substitution (Tyrxe2x86x92His) at the polypeptide level. A mutation in exon 5 introduces a stop codon into the ORF leading to the expression of a truncated MGST-II polypeptide. These mutations modify the specificity, activity and function of the MGST-II enzyme and therefore represent functional mutations of the MGST-II gene. Candidate polymorphisms and mutations suspected of being responsible for the detectable phenotype, such as hepatotoxicity to zileuton or asthma, can be confirmed by screening a larger population of affected and unaffected individuals using any of the genotyping procedures described herein. Preferably the microsequencing technique is used. In a preferred embodiment trait positive and trait negative populations are genotyped for the candidate polymorphisms identified in Example 5 (10-286-289, 10-286-345, 10-286-375, 10-523-232, 10-289-201, 10-290-37, 10-290-326 and 10-290-328) most preferably for the mutations in exons 4 and 5 (10-289-201 and 10-290-37). Polymorphisms are considered as candidate xe2x80x9ctrait-causingxe2x80x9d mutations when they exhibit a statistically significant correlation with the detectable phenotype.
V. Biallelic Markers of the Invention in Methods of Genetic Diagnostics
The biallelic markers of the present invention can also be used to develop diagnostics tests capable of identifying individuals who express a detectable trait as the result of a specific genotype or individuals whose genotype places them at risk of developing a detectable trait at a subsequent time. The trait analyzed using the present diagnostics may be any detectable trait, including a response to a drug or side effects to a drug upon treatment or a disease involving the metabolic conversion of xenobiotics.
The diagnostic techniques of the present invention may employ a variety of methodologies to determine whether a test subject has a biallelic marker pattern associated with an increased risk of developing a detectable trait or whether the individual suffers from a detectable trait as a result of a particular mutation, including methods which enable the analysis of individual chromosomes for haplotyping, such as family studies, single sperm DNA analysis or somatic hybrids.
The present invention provides diagnostic methods to determine whether an individual is at risk of developing a disease or suffers from a disease resulting from a mutation or a polymorphism in a candidate gene of the present invention. The present invention also provides methods to determine whether an individual is likely to respond positively to a therapeutic agent or whether an individual is at risk of developing an adverse side effect to a therapeutic agent.
These methods involve obtaining a nucleic acid sample from the individual and, determining, whether the nucleic acid sample contains at least one allele or at least one biallelic marker haplotype, indicative of a risk of developing the trait or indicative that the individual expresses the trait as a result of possessing a particular candidate gene polymorphism or mutation (trait-causing allele).
Preferably, in such diagnostic methods, a nucleic acid sample is obtained from the individual and this sample is genotyped using methods described herein in xe2x80x9cMethods of Genotyping an Individual for Biallelic Markers.xe2x80x9d The diagnostics may be based on a single biallelic marker or on a group of biallelic markers.
In each of these methods, a nucleic acid sample is obtained from the test subject and the biallelic marker pattern of one or more of the biallelic markers listed in Table 11(A-B) is determined.
In one embodiment, a PCR amplification is conducted on the nucleic acid sample to amplify regions in which polymorphisms associated with a detectable phenotype have been identified. The amplification products are sequenced to determine whether the individual possesses one or more polymorphisms associated with a detectable phenotype. The primers used to generate amplification products may comprise the primers listed in Table 17. Alternatively, the nucleic acid sample is subjected to microsequencing reactions as described above to determine whether the individual possesses one or more polymorphisms associated with a detectable phenotype resulting from a mutation or a polymorphism in a candidate gene. The primers used in the microsequencing reactions may include the primers listed in Table 16. In another embodiment, the nucleic acid sample is contacted with one or more allele specific oligonucleotide probes which, specifically hybridize to one or more candidate gene alleles associated with a detectable phenotype. The probes used in the hybridization assay may include the probes listed in Table 18. Diagnostic kits comprising polynucleotides of the present invention are further described in section I.
These diagnostic methods are extremely valuable as they can, in certain circumstances, be used to initiate preventive treatments or to allow an individual carrying a significant haplotype to foresee warning signs such as minor symptoms. For diseases in which attacks may be extremely violent and sometimes fatal if not treated on time, the knowledge of a potential predisposition, even if this predisposition is not absolute, might contribute in a very significant manner to treatment efficacy. Similarly, a diagnosed predisposition to a potential side effect could immediately direct the physician toward a treatment for which such side effects have not been observed during clinical trials.
Diagnostics, which analyze and predict response to a drug or side effects to a drug, may be used to determine whether an individual should be treated with a particular drug. For example, if the diagnostic indicates a likelihood that an individual will respond positively to treatment with a particular drug, the drug may be administered to the individual. Conversely, if the diagnostic indicates that an individual is likely to respond negatively to treatment with a particular drug, an alternative course of treatment may be prescribed. A negative response may be defined as either the absence of an efficacious response or the presence of toxic side effects.
Clinical drug trials represent another application for the markers of the present invention. One or more markers indicative of response to a drug or to side effects to a drug may be identified using the methods described above. Thereafter, potential participants in clinical trials of such an agent may be screened to identify those individuals most likely to respond favorably to the drug and exclude those likely to experience side effects. In that way, the effectiveness of drug treatment may be measured in individuals who respond positively to the drug, without lowering the measurement as a result of the inclusion of individuals who are unlikely to respond positively in the study and without risking undesirable safety problems.
In a preferred embodiment the identity of the nucleotide present at, at least one, biallelic marker selected from the group consisting of 12-455-326, 12-453-429, 12-454-363, 12-441-233, 12-461-299, 12-426-154, 12-424-198, 12-716-295, 10-428-219, 12-720-80, 12-156-91, 12-140-134, 12-653-423, 10-471-84, 10-471-85, 10-470-25, 12-652-203, 12-637-219, 12-721-440 and 10-420-284 is determined, and optionally wherein the detectable trait is asthma, or optionally the detectable trait is hepatotoxicity to the anti-asthmatic drug zileuton. In another preferred embodiment the identity of the nucleotide present at, at least one of the polymorphic sites selected from the group consisting of 12-447-58, 12-455-326, 12-461-299, 12-453-429, 12-424-198, 12-454-363, 12-716-295, 10-428-219, 12-720-80, 10-420-248, 12-721-440, 12-653-423, 10-470-25, 10-471-84, 10-471-85, 12-637-219 and 12-652-203 is determined, and optionally wherein the detectable trait is asthma. In another preferred embodiment the identity of the nucleotide present at, at least one of the polymorphic sites selected from the group consisting of 12-453-429, 12-455-326, 12-453-429, 12-454-363, 12-441-233, 12-461-299, 12-426-154, 12-424-198, 12-716-295, 10-428-219, 12-720-80, 12-156-91, 12-140-134, 12-653-423, 10-471-84, 10-471-85, 10-470-25, 12-652-203, 12-637-219, 12-721-440, and 10-420-284 is determined, and optionally wherein the detectable trait is hepatotoxicity to the anti-asthmatic drug zileuton. Diagnostic kits comprising polynucleotides of the present invention are further described in xe2x80x9cBiallelic Markers and Polynucleotides Comprising Biallelic Markers.xe2x80x9d
VI. Association of Biallelic Markers of the Invention with Asthma
In the context of the present invention, an association between the MGST-II, ME1, UGT1A7 and UGT2B4 genes and asthma was established. See Example 3.
Asthma affects over 5% of the population in industrialized countries. It is increasing in prevalence and severity and has a rising mortality (Rang H. P., Ritter J. M. and Dale M. M.; Pharmacology; Churchill Livingstone, N.Y., 1995). Bronchial asthma is a multifactorial syndrome rather than a single disease, defined as airway obstruction characterized by inflammatory changes in the airways and bronchial hyper-responsiveness. In addition to the evidenced impact of environmental factors on the development of asthma, patterns of clustering and segregation in asthmatic families have suggested a genetic component to asthma. However the lack of a defined and specific asthma phenotype and of suitable markers for genetic analysis is proving to be a major hurdle for reliably identifying genes associated with asthma. The identification of genes implicated in asthma would represent a major step towards the identification of new molecular targets for the development of anti-asthma drugs. Moreover there is no straightforward physiological or biological blood test for the asthmatic state. As a result, adequate asthma treatment is often delayed, thereby allowing the inflammation process to better establish itself. Thus, there is a need for the identification of asthma susceptibility genes in order to develop an efficient and reliable asthma diagnostic test.
As mentioned above, products of arachidonic acid metabolism are important inflammatory mediators and have been involved in a number of inflammatory diseases, including asthma. More specifically, prostaglandins and leukotrienes are thought to play a major role in the inflammatory process observed in asthma patients.
In order to investigate and identify a genetic origin to asthma, a candidate gene scan was conducted. This approach comprised:
a) selecting candidate genes potentially involved in the pathological pathway of interest, in this case arachidonic acid metabolism, and
b) identifying biallelic markers in those genes, and finally
c) conducting association studies to identify biallelic marker alleles or haplotypes associated with asthma.
Further details concerning this association study are provided in Example 3, results are briefly summarized below.
Two groups of independent individuals were used in this association study in accordance with the invention: the case-control populations. The two groups corresponded to 297 asthmatic individuals and 178 control individuals. The trait positive asthma population was mostly composed of individuals from Caucasian ethnic background ( greater than 90%). The control population was composed of individuals from a random US Caucasian population.
In the association study described in Example 3, several biallelic marker haplotypes were shown to be significantly associated with asthma. A preferred haplotype consisting of three biallelic markers (12-455-326, 12-453-429 and 12-424-198) presented a p-value of 3.2 10xe2x88x925. Another preferred haplotype consisting of four biallelic markers (12-455-326, 12-453-429, 12-424-198 and 12-454-363) had a p-value of 1.2 10xe2x88x926. Phenotypic permutation tests confirmed the statistical significance of these results. These haplotypes can therefore be considered to be significantly associated with asthma.
This information is extremely valuable. The knowledge of a potential genetic predisposition to asthma, even if this predisposition is not absolute, might contribute in a very significant manner to treatment efficacy of asthma patients and to the development of new therapeutic and diagnostic tools.
VII. Association of Biallelic Markers of the Invention with Hepatotoxicity to Anti-Asthma Drug Zileuton (Zyflo(trademark))
In the context of the present invention, an association between the the MGST-II, ME1, UGT1A7 and UGT2B4 genes and side effects related to treatment with the anti-asthmatic drug zileuton was discovered.
As mentioned above, bronchial asthma is a multifactorial syndrome rather than a single disease, defined as airway obstruction characterized by inflammatory changes in the airways and bronchial hyper-responsiveness. Although initially reversible with bronchiodilators, airway obstruction becomes increasingly irreversible if treated poorly. Asthma management therefore relies on early and regular use of drugs that control the disease. As a consequence, there is a strong need for efficient and safe therapeutic opportunities for patients with asthma. There are two main categories of anti-asthmatic drugsxe2x80x94bronchodilators and anti-inflammatory agents. There is now general agreement on the need to implement early anti-inflammatory treatment rather than relying on symptomatic treatment with bronchiodilators alone. The leukotrienes, a family of proinflammatory mediators arising via arachidonic acid metabolism, have been implicated in the inflammatory cascade that occurs in asthmatic airways. Of great relevance to the pathogenesis of asthma are the 5-lipoxygenase and the 5-lipoxygenase activating protein (FLAP), which catalyze the initial steps in the biosynthesis of leukotrienes from arachidonic acid. Given the significant role of the inflammatory process in asthma, pharmacological agents, such as leukotriene antagonists, FLAP inhibitors and 5-lipoxygenase inhibitors have been developed.
Zileuton (Zyflo(trademark)) is an active inhibitor of 5-lipoxygenase, the enzyme that catalyzes the formation of leukotrienes from arachidonic acid, indicated for prophylaxis and chronic treatment of asthma. A minority of zileuton-treated patients develop liver function abnormalities as close monitoring revealed that elevations of liver function tests may occur during treatment with zileuton. In the present invention the ALT test (serum level of alanine aminotransferase) was used, which is considered the most sensitive indicator of liver injury.
In order to investigate and identify a genetic origin to zileuton-associated hepatotoxicity, a candidate gene scan was conducted. This approach comprised:
a) selecting candidate genes potentially involved in the pathological pathway of interest or in the metabolism of zileuton, and
b) identifying biallelic markers in those genes, and finally
c) conducting association studies to identify biallelic marker alleles or haplotypes associated with elevations of liver function tests upon treatment with zileuton.
Further details concerning this association study are provided in Example 4, results are briefly summarized below.
Two groups of unrelated individuals were used in this association study in accordance with the invention: the case-control populations. The case population was composed of 89 asthmatic individuals treated with zileuton showing zileuton-associated hepatotoxicity monitored by the serum level of alanine aminotransferase (ALT) and the control population was composed of 208 asthmatic individuals treated with zileuton and having no increased serum ALT level.
The association study conducted with the biallelic markers derived from the MGST-II locus showed that several haplotypes were significantly associated with zileuton-associated hepatotoxicity. A preferred haplotype consisting of three biallelic markers (12-441-233, 12-461-299 and 12-453-429) presented a p-value of 1.5 10xe2x88x925 and an odd ratio of 3.63. A second preferred haplotype consisting of four biallelic markers (12-441-233, 12-461-299, 12-453-429 and 12-426-154) had a p-value of 5.2 10xe2x88x927 and an odd ratio of 5.75.
This information is extremely valuable. The knowledge of a potential genetic predisposition to hepatotoxicity upon treatment with zileuton, even if this predisposition is not absolute, might contribute in a very significant manner to the safety of asthma treatment and to the development of diagnostic tools.
Similar association studies, with different case-control populations, can be routinely carried out by the skilled technician using the biallelic markers of the present invention in order to identify other association between traits and DME and MGST-II-related biallelic marker alleles or haplotypes.
VI. Computer-Related Embodiments
As used herein the term xe2x80x9cnucleic acid codes of the inventionxe2x80x9d encompass the nucleotide sequences comprising, consisting essentially of, or consisting of any one of the following: a) a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500 or 1000 nucleotides, to the extent that a polynucleotide of these lengths is consistent with the lengths of the particular Sequence ID, of a sequence selected from the group consisting of the sequences described in Table 12, and the complements thereof; b) a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500 or 1000 nucleotides, to the extent that a polynucleotide of these lengths is consistent with the lengths of the particular Sequence ID, of a sequence selected from the group consisting of the sequences described in Table 13, and the complements thereof, c) a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, or 500 nucleotides, to the extent that a polynucleotide of these lengths is consistent with the lengths of the particular Sequence ID, of a sequence selected from the group consisting of the sequences described in Table 16, more preferably a set of markers or sequences consisting of those markers or sequences found in SEQ ID Nos. 3, 5, 9, 13-15, 25, 31, 33, 37, 38, 41, 323, 345, 351-353, 357, 377, and 380, and the complements thereof, wherein said span includes an DME-related biallelic marker, preferably an DME-related biallelic marker described in Table 1 l(A-B), preferably the biallelic markers found in Tables 19, 20, 21 and 22; or more preferably the biallelic markers found in SEQ ID Nos. 3, 5, 9, 13-15, 25, 31, 33, 37, 38, 41, 323, 345, 351-353, 357, 377, 380, in said sequence with the alternative allele present at said biallelic marker.
The xe2x80x9cnucleic acid codes of the inventionxe2x80x9d further encompass nucleotide sequences homologous to a contiguous span of at least 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500 or 1000 nucleotides, to the extent that a contiguous span of these lengths is consistent with the lengths of the particular Sequence ID, of a sequence selected from the group consisting of the sequences described in Tables 12, 13 and 16 and the complements thereof. Homologous sequences refer to a sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, 80%, or 75% homology to these contiguous spans. Homology may be determined using any method described herein, including BLAST2N with the default parameters or with any modified parameters. Homologous sequences also may include RNA sequences in which uridines replace the thymines in the nucleic acid codes of the invention. It will be appreciated that the nucleic acid codes of the invention can be represented in the traditional single character format (See the inside back cover of Stryer, Lubert. Biochemistry, 3rd edition. W. H Freeman and Co., New York.) or in any other format or code which records the identity of the nucleotides in a sequence.
It will be appreciated by those skilled in the art that the nucleic acid codes of the invention, one or more of the polypeptide codes of SEQ ID Nos. 488 and 489 can be stored, recorded, and manipulated on any medium which can be read and accessed by a computer. As used herein, the words xe2x80x9crecordedxe2x80x9d and xe2x80x9cstoredxe2x80x9d refer to a process for storing information on a computer medium. A skilled artisan can readily adopt any of the presently known methods for recording information on a computer readable medium to generate manufactures comprising one or more of the nucleic acid codes of the invention and one or more of the polypeptide codes of SEQ ID Nos. 488 and 489. Another aspect of the present invention is a computer readable medium having recorded thereon at least 2, 5, 10, 15, 20, 25, 30, or 50 nucleic acid codes of the invention, and the complements thereof. Another aspect of the present invention is a computer readable medium having recorded thereon at least 2, 5, 10, 15, 20, 25, 30, or 50 polypeptide codes of SEQ ID Nos. 488 and 489.
Computer readable media include magnetically readable media, optically readable media, electronically readable media and magnetic/optical media. For example, the computer readable media may be a hard disk, a floppy disk, a magnetic tape, CD-ROM, Digital Versatile Disk (DVD), Random Access Memory (RAM), or Read Only Memory (ROM) as well as other types of other media known to those skilled in the art.
Embodiments of the present invention include systems, particularly computer systems which store and manipulate the sequence information described herein. As used herein, xe2x80x9ca computer systemxe2x80x9d refers to the hardware components, software components, and data storage components used to analyze the nucleotide sequences of the nucleic acid codes of the invention , or the amino acid sequences of the polypeptide codes of SEQ ID Nos. 488 and 489. In one embodiment, the computer system is a Sun Enterprise 1000 server (Sun Microsystems, Palo Alto, Calif.). The computer system preferably includes a processor for processing, accessing and manipulating the sequence data. The processor can be any well-known type of central processing unit, such as the Pentium III from Intel Corporation, or similar processor from Sun, Motorola, Compaq or International Business Machines. Preferably, the computer system is a general-purpose system that comprises the processor and one or more internal data storage components for storing data, and one or more data retrieving devices for retrieving the data stored on the data storage components. A skilled artisan can readily appreciate that any one of the currently available computer systems are suitable. In one particular embodiment, the computer system includes a processor connected to a bus which is connected to a main memory (preferably implemented as RAM) and one or more internal data storage devices, such as a hard drive and/or other computer readable media having data recorded thereon. In some embodiments, the computer system further includes one or more data-retrieving device for reading the data stored on the internal data storage devices. The data-retrieving device may represent, for example, a floppy disk drive, a compact disk drive, a magnetic tape drive, etc. In some embodiments, the internal data storage device is a removable computer readable medium such as a floppy disk, a compact disk, a magnetic tape, etc. containing control logic and/or data recorded thereon. The computer system may advantageously include or be programmed by appropriate software for reading the control logic and/or the data from the data storage component once inserted in the data-retrieving device. The computer system includes a display which is used to display output to a computer user. It should also be noted that the computer system can be linked to other computer systems in a network or wide area network to provide centralized access to the computer system. Software for accessing and processing the nucleotide sequences of the nucleic acid codes of the invention, or the amino acid sequences of the polypeptide codes of SEQ ID Nos. 488 and 489 (such as search tools, compare tools, and modeling tools etc.) may reside in main memory during execution. In some embodiments, the computer system may further comprise a sequence comparer for comparing the above-described nucleic acid codes of the invention or polypeptide codes of SEQ ID Nos. 488 and 489 stored on a computer readable medium to reference nucleotide or polypeptide sequences stored on a computer readable medium. A xe2x80x9csequence comparerxe2x80x9d refers to one or more programs which are implemented on the computer system to compare a nucleotide or polypeptide sequence with other nucleotide or polypeptide sequences and/or compounds including but not limited to peptides, peptidomimetics, and chemicals stored within the data storage means. For example, the sequence comparer may compare the nucleotide sequences of the nucleic acid codes of the invention, or the amino acid sequences of the polypeptide codes of SEQ ID Nos. 488 and 489 stored on a computer readable medium to reference sequences stored on a computer readable medium to identify homologies, motifs implicated in biological function, or structural motifs. The various sequence comparer programs identified elsewhere in this patent specification are particularly contemplated for use in this aspect of the invention.
One embodiment is a process for comparing a new nucleotide or protein sequence with a database of sequences in order to determine the homology levels between the new sequence and the sequences in the database. The database of sequences can be a private database stored within the computer system, or a public database such as GENBANK, PIR OR SWISSPROT that is available through the Internet.
The process begins at a start state and then moves to a state wherein the new sequence to be compared is stored to a memory in a computer system. As discussed above, the memory could be any type of memory, including RAM or an internal storage device. The process then moves to a state wherein a database of sequences is opened for analysis and comparison. The process then moves to a state wherein the first sequence stored in the database is read into a memory on the computer. A comparison is then performed at a state to determine if the first sequence is the same as the second sequence. It is important to note that this step is not limited to performing an exact comparison between the new sequence and the first sequence in the database. Well-known methods are known to those of skill in the art for comparing two nucleotide or protein sequences, even if they are not identical. For example, gaps can be introduced into one sequence in order to raise the homology level between the two tested sequences. The parameters that control whether gaps or other features are introduced into a sequence during comparison are normally entered by the user of the computer system. Once a comparison of the two sequences has been performed at the state, a determination is made at a decision state whether the two sequences are the same. Of course, the term xe2x80x9csamexe2x80x9d is not limited to sequences that are absolutely identical. Sequences that are within the homology parameters entered by the user will be marked as xe2x80x9csamexe2x80x9d in the process. If a determination is made that the two sequences are the same, the process moves to a state wherein the name of the sequence from the database is displayed to the user. This state notifies the user that the sequence with the displayed name fulfills the homology constraints that were entered. Once the name of the stored sequence is displayed to the user, the process moves to a decision state wherein a determination is made whether more sequences exist in the database. If no more sequences exist in the database, then the process terminates at an end state. However, if more sequences do exist in the database, then the process moves to a state wherein a pointer is moved to the next sequence in the database so that it can be compared to the new sequence. In this manner, the new sequence is aligned and compared with every sequence in the database. It should be noted that if a determination had been made at the decision state that the sequences were not homologous, then the process would move immediately to the decision state in order to determine if any other sequences were available in the database for comparison. Accordingly, one aspect of the present invention is a computer system comprising a processor, a data storage device having stored thereon a nucleic acid code of the invention or a polypeptide code of SEQ ID Nos. 488 and 489, a data storage device having retrievably stored thereon reference nucleotide sequences or polypeptide sequences to be compared to the nucleic acid code of the invention or polypeptide code of SEQ ID Nos. 488 and 489 and a sequence comparer for conducting the comparison. The sequence comparer may indicate a homology level between the sequences compared or identify structural motifs in the above described nucleic acid code of the invention and polypeptide codes of SEQ ID Nos. 488 and 489 or it may identify structural motifs in sequences which are compared to these nucleic acid codes and polypeptide codes. In some embodiments, the data storage device may have stored thereon the sequences of at least 2, 5, 10, 15, 20, 25, 30, or 50 of the nucleic acid codes of the invention or polypeptide codes of SEQ ID Nos. 488 and 489.
Another aspect of the present invention is a method for determining the level of homology between a nucleic acid code of the invention and a reference nucleotide sequence, comprising the steps of reading the nucleic acid code and the reference nucleotide sequence through the use of a computer program which determines homology levels and determining homology between the nucleic acid code and the reference nucleotide sequence with the computer program. The computer program may be any of a number of computer programs for determining homology levels, including those specifically enumerated herein, including BLAST2N with the default parameters or with any modified parameters. The method may be implemented using the computer systems described above. The method may also be performed by reading 2, 5, 10, 15, 20, 25, 30, or 50 of the above described nucleic acid codes of the invention through use of the computer program and determining homology between the nucleic acid codes and reference nucleotide sequences .
One embodiment is a process in a computer for determining whether two sequences are homologous. The process begins at a start state and then moves to a state wherein a first sequence to be compared is stored to a memory. The second sequence to be compared is then stored to a memory at a state. The process then moves to a state wherein the first character in the first sequence is read and then to a state wherein the first character of the second sequence is read. It should be understood that if the sequence is a nucleotide sequence, then the character would normally be either A, T, C, G or U. If the sequence is a protein sequence, then it should be in the single letter amino acid code so that the first and sequence sequences can be easily compared.
A determination is then made at a decision state whether the two characters are the same. If they are the same, then the process moves to a state wherein the next characters in the first and second sequences are read. A determination is then made whether the next characters are the same. If they are, then the process continues this loop until two characters are not the same. If a determination is made that the next two characters are not the same, the process moves to a decision state to determine whether there are any more characters either sequence to read. If there aren""t any more characters to read, then the process moves to a state wherein the level of homology between the first and second sequences is displayed to the user. The level of homology is determined by calculating the proportion of characters between the sequences that were the same out of the total number of sequences in the first sequence. Thus, if every character in a first 100 nucleotide sequence aligned with a every character in a second sequence, the homology level would be 100%. Alternatively, the computer program may be a computer program which compares the nucleotide sequences of the nucleic acid codes of the present invention, to reference nucleotide sequences in order to determine whether the nucleic acid code of SEQ ID Nos. 1-977 differs from a reference nucleic acid sequence at one or more positions. Optionally such a program records the length and identity of inserted, deleted or substituted nucleotides with respect to the sequence of either the reference polynucleotide or the nucleic acid code of SEQ ID Nos. 1-977. In one embodiment, the computer program may be a program which determines whether the nucleotide sequences of the nucleic acid codes of the invention contain a biallelic marker or single nucleotide polymorphism (SNP) with respect to a reference nucleotide sequence. This single nucleotide polymorphism may comprise a single base substitution, insertion, or deletion, while this biallelic marker may comprise about one to ten consecutive bases substituted, inserted or deleted.
Another aspect of the present invention is a method for determining the level of homology between a polypeptide code of SEQ ID Nos. 488 and 489 and a reference polypeptide sequence, comprising the steps of reading the polypeptide code of SEQ ID Nos. 488 and 489 and the reference polypeptide sequence through use of a computer program which determines homology levels and determining homology between the polypeptide code and the reference polypeptide sequence using the computer program.
Accordingly, another aspect of the present invention is a method for determining whether a nucleic acid code of the invention differs at one or more nucleotides from a reference nucleotide sequence comprising the steps of reading the nucleic acid code and the reference nucleotide sequence through use of a computer program which identifies differences between nucleic acid sequences and identifying differences between the nucleic acid code and the reference nucleotide sequence with the computer program. In some embodiments, the computer program is a program which identifies single nucleotide polymorphisms. The method may be implemented by the computer systems described above. The method may also be performed by reading at least 2, 5, 10, 15, 20, 25, 30, or 50 of the nucleic acid codes of the invention and the reference nucleotide sequences through the use of the computer program and identifying differences between the nucleic acid codes and the reference nucleotide sequences with the computer program. In other embodiments the computer based system may further comprise an identifier for identifying features within the nucleotide sequences of the nucleic acid codes of the invention or the amino acid sequences of the polypeptide codes of SEQ ID Nos. 488 and 489. An xe2x80x9cidentifierxe2x80x9d refers to one or more programs which identifies certain features within the above-described nucleotide sequences of the nucleic acid codes of the invention or the amino acid sequences of the polypeptide codes of SEQ ID Nos. 488 and 489. In one embodiment, the identifier may comprise a program which identifies an open reading frame in the cDNAs codes of SEQ ID Nos. 486 and 487.
One embodiment is an identifier process for detecting the presence of a feature in a sequence. The process begins at a start state and then moves to a state wherein a first sequence that is to be checked for features is stored to a memory in the computer system. The process then moves to a state wherein a database of sequence features is opened. Such a database would include a list of each feature""s attributes along with the name of the feature. For example, a feature name could be xe2x80x9cInitiation Codonxe2x80x9d and the attribute would be xe2x80x9cATG.xe2x80x9d Another example would be the feature name xe2x80x9cTAATAA Boxxe2x80x9d and the feature attribute would be xe2x80x9cTAATAAxe2x80x9d. An example of such a database is produced by the University of Wisconsin Genetics Computer Group (world wide web site: gcg.com). Once the database of features is opened at the state, the process moves to a state wherein the first feature is read from the database. A comparison of the attribute of the first feature with the first sequence is then made at a state. A determination is then made at a decision state whether the attribute of the feature was found in the first sequence. If the attribute was found, then the process moves to a state wherein the name of the found feature is displayed to the user. The process then moves to a decision state wherein a determination is made whether move features exist in the database. If no more features do exist, then the process terminates at an end state. However, if more features do exist in the database, then the process reads the next sequence feature at a state and loops back to the state wherein the attribute of the next feature is compared against the first sequence. It should be noted, that if the feature attribute is not found in the first sequence at the decision state, the process moves directly to the decision state in order to determine if any more features exist in the database. In another embodiment, the identifier may comprise a molecular modeling program which determines the 3-dimensional structure of the polypeptides codes of SEQ ID Nos. 488 and 489. In some embodiments, the molecular modeling program identifies target sequences that are most compatible with profiles representing the structural environments of the residues in known three-dimensional protein structures. (See, e.g., Eisenberg et al., U.S. Pat. No. 5,436,850 issued Jul. 25, 1995, the disclosure of which is incorporated herein by reference in its entirety). In another technique, the known three-dimensional structures of proteins in a given family are superimposed to define the structurally conserved regions in that family. This protein modeling technique also uses the known three-dimensional structure of a homologous protein to approximate the structure of the polypeptide codes of SEQ ID Nos. 488 and 489. (See e.g., Srinivasan, et al., U.S. Pat. No. 5,557,535 issued Sep. 17, 1996, the disclosure of which is incorporated herein by reference in its entirety). Conventional homology modeling techniques have been used routinely to build models of proteases and antibodies. (Sowdhamini et al., Protein Engineering 10:207, 215 (1997), the disclosure of which is incorporated herein by reference in its entirety). Comparative approaches can also be used to develop three-dimensional protein models when the protein of interest has poor sequence identity to template proteins. In some cases, proteins fold into similar three-dimensional structures despite having very weak sequence identities. For example, the three-dimensional structures of a number of helical cytokines fold in similar three-dimensional topology in spite of weak sequence homology. The recent development of threading methods now enables the identification of likely folding patterns in a number of situations where the structural relatedness between target and template(s) is not detectable at the sequence level. Hybrid methods, in which fold recognition is performed using Multiple Sequence Threading (MST), structural equivalencies are deduced from the threading output using a distance geometry program DRAGON to construct a low resolution model, and a full-atom representation is constructed using a molecular modeling package such as QUANTA.
According to this 3-step approach, candidate templates are first identified by using the novel fold recognition algorithm MST, which is capable of performing simultaneous threading of multiple aligned sequences onto one or more 3-D structures. In a second step, the structural equivalencies obtained from the MST output are converted into interresidue distance restraints and fed into the distance geometry program DRAGON, together with auxiliary information obtained from secondary structure predictions. The program combines the restraints in an unbiased manner and rapidly generates a large number of low resolution model confirmations. In a third step, these low resolution model confirmations are converted into full-atom models and subjected to energy minimization using the molecular modeling package QUANTA. (See e.g., Aszxc3x3di et al., Proteins: Structure, Function, and Genetics, Supplement 1:38-42 (1997), the disclosure of which is incorporated herein by reference in its entirety).
The results of the molecular modeling analysis may then be used in rational drug design techniques to identify agents which modulate the activity of the polypeptide codes of SEQ ID Nos. 488 and 489. Accordingly, another aspect of the present invention is a method of identifying a feature within the nucleic acid codes of the invention or the polypeptide codes of SEQ ID Nos. 488 and 489 comprising reading the nucleic acid code(s) or the polypeptide code(s) through the use of a computer program which identifies features therein and identifying features within the nucleic acid code(s) or polypeptide code(s) with the computer program. In one embodiment, computer program comprises a computer program which identifies open reading frames. In a further embodiment, the computer program identifies structural motifs in a polypeptide sequence. In another embodiment, the computer program comprises a molecular modeling program. The method may be performed by reading a single sequence or at least 2, 5, 10, 15, 20, 25, 30, or 50 of the nucleic acid codes of the invention or the polypeptide codes of SEQ ID Nos. 488 and 489 through the use of the computer program and identifying features within the nucleic acid codes or polypeptide codes with the computer program. The nucleic acid codes of the invention or the polypeptide codes of SEQ ID Nos. 488 and 489 may be stored and manipulated in a variety of data processor programs in a variety of formats. For example, the nucleic acid codes of the invention or the polypeptide codes of SEQ ID Nos. 488 and 489 may be stored as text in a word processing file, such as MicrosoftWORD or WORDPERFECT or as an ASCII file in a variety of database programs familiar to those of skill in the art, such as DB2, SYBASE, or ORACLE. In addition, many computer programs and databases may be used as sequence comparers, identifiers, or sources of reference nucleotide or polypeptide sequences to be compared to the nucleic acid codes of the invention or the polypeptide codes of SEQ ID Nos. 488 and 489. The following list is intended not to limit the invention but to provide guidance to programs and databases which are useful with the nucleic acid codes of the invention or the polypeptide codes of SEQ ID No. 488 and 489. The programs and databases which may be used include, but are not limited to: MacPattern (EMBL), DiscoveryBase (Molecular Applications Group), GeneMine (Molecular Applications Group), Look (Molecular Applications Group), MacLook (Molecular Applications Group), BLAST and BLAST2 (NCBI), BLASTN and BLASTX (Altschul et al, J. Mol. Biol. 215: 403 (1990), the disclosure of which is incorporated herein by reference in its entirety), FASTA (Pearson and Lipman, Proc. Natl. Acad. Sci. USA, 85: 2444 (1988), the disclosure of which is incorporated herein by reference in its entirety), FASTDB (Brutlag et al. Comp. App. Biosci. 6:237-245, 1990, the disclosure of which is incorporated herein by reference in its entirety), Catalyst (Molecular Simulations Inc.), Catalyst/SHAPE (Molecular Simulations Inc.), Cerius2.DBAccess (Molecular Simulations Inc.), HypoGen (Molecular Simulations Inc.), Insight II, (Molecular Simulations Inc.), Discover (Molecular Simulations Inc.), CHARMm (Molecular Simulations Inc.), Felix (Molecular Simulations Inc.), DelPhi, (Molecular Simulations Inc.), QuanteMM, (Molecular Simulations Inc.), Homology (Molecular Simulations Inc.), Modeler (Molecular Simulations Inc.), ISIS (Molecular Simulations Inc.), Quanta/Protein Design (Molecular Simulations Inc.), WebLab (Molecular Simulations Inc.), WebLab Diversity Explorer (Molecular Simulations Inc.), Gene Explorer Molecular Simulations Inc.), SeqFold (Molecular Simulations Inc.), the EMBL/Swissprotein database, the MDL Available Chemicals Directory database, the MDL Drug Data Report data base, the Comprehensive Medicinal Chemistry database, Derwents""s World Drug Index database, the BioByteMasterFile database, the Genbank database, and the Genseqn database. Many other programs and data bases would be apparent to one of skill in the art given the present disclosure. Motifs which may be detected using the above programs include sequences encoding leucine zippers, helix-turn-helix motifs, glycosylation sites, ubiquitination sites, alpha helices, and beta sheets, signal sequences encoding signal peptides which direct the secretion of the encoded proteins, sequences implicated in transcription regulation such as homeoboxes, acidic stretches, enzymatic active sites, substrate binding sites, and enzymatic cleavage sites.
It should be noted that the nucleic acid codes of the invention further encompass all of the polynucleotides disclosed, described or claimed in the present invention. Also, it should be noted that the polypeptide codes of SEQ ID Nos. 488 and 489 further encompass all of the polypeptides disclosed, described or claimed in the present invention. Moreover, the present invention specifically contemplates the storage of such codes on computer readable media and computer systems individually or in combination, as well as the use of such codes and combinations in the methods of section xe2x80x9cVI. Computer-Related Embodiments.xe2x80x9d
Throughout this application, various publications, patents, and published patent applications are cited. The disclosures of the publications, patents, and published patent specifications referenced in this application are hereby incorporated by reference into the present disclosure to more fully describe the state of the art to which this invention pertains.
VII. DNA Typing Methods and Systems
The present invention also encompasses a DNA typing system having a much higher discriminatory power than currently available typing systems. The systems and associated methods are particularly applicable in the identification of individuals for forensic science and paternity determinations. These applications have become increasingly important; in forensic science, for example, the identification of individuals by polymorphism analysis has become widely accepted by courts as evidence.
While forensic geneticists have developed many techniques to compare homologous segments of DNA to determine if the segments are identical or if they differ in one or more nucleotides, each technique still has certain disadvantages. In particular, the techniques vary widely in terms of expense of analysis, time required to carry out an analysis and statistical power.
RFLP Analysis Methods
The best known and most widespread method in forensic DNA typing is the restriction fragment length polymorphism (RFLP) analysis. In RFLP testing, a repetitive DNA sequence referred to as a variable number tandem repeat (VNTR) which varies between individuals is analyzed. The core repeat is typically a sequence of about 15 base pairs in length, and highly polymorphic VNTR loci can have an average of about 20 alleles. DNA restriction sites located on either site of the VNTR are exploited to create DNA fragments from about 0.5 Kb to less than 10 Kb which are then separated by electrophoresis, indicating the number of repeats found in the individual at the particular loci. RFLP methods generally consist of (1) extraction and isolation of DNA, (2) restriction endonuclease digestion; (3) separation of DNA fragments by electrophoresis; (4) capillary transfer; (5) hybridization with radiolabelled probes; (6) autoradiography; and (7) interpretation of results (Lee, H. C. et al., Am. J. Forensic. Med. Pathol. 15(4): 269-282 (1994)). RFLP methods generally combine analysis at about 5 loci and have much higher discriminate potential than other available test due the highly polymorphic nature of the VNTRs. However, autoradiography is costly and time consuming and an analysis generally takes weeks or months for turnaround. Additionally, a large amount of sample DNA is required, which is often not available at a crime scene. Furthermore, the reliability of the system and its credibility as evidence is decreased because the analysis of tightly spaced bands on electrophoresis results in a high rate of error.
PCR Methods
PCR based methods offer an alternative to RFLP methods. In a first method called AmpFLP, DNA fragments containing VNTRs are amplified and then separated electrophoretically, without the restriction step of RFLP method. While this method allows small quantities of sample DNA to be used, decreases analysis time by avoiding autoradiography, and retains high discriminatory potential, it nevertheless requires electrophoretic separation which takes substantial time and introduces an significant error rate. In another AmpFLP method, short tandem repeats (STRs) of 2 to 8 base pairs are analyzed. STRs are more suitable to analysis of degraded DNA samples since they require smaller amplified fragments but have the disadvantage of requiring separation of the amplified fragments. While STRs are far less informative than longer repeats, similar discriminatory potential can be achieved if enough STRs are used in a single analysis.
Other methods include sequencing of mitochondrial DNA, which is especially suitable for situations where sample DNA is very degraded or in small quantities. However, only a small region of 1 Kb of the mitochondrial DNA referred to as the D-Loop locus has been found useful for typing because of its polymorphic nature, resulting in lower discriminatory potential than with RFLP or AmpFLP methods. Furthermore, DNA sequencing is expensive to carry out on a large number of samples.
Further available methods include dot-blot methods, which involve using allele specific oligonucleotide probes which hybridize sequence specifically to one allele of a polymorphic site. Systems include the HLA DQ-alpha kit developed by Cetus Corp. which has a discriminatory value of about 1 in 20, and a dot-blot strip referred to as the Polymarker strip combining five genetic loci for a discriminatory value of about one in a few thousand. (Weedn, V., Clinics in Lab. Med. 16(1): 187-196 (1996)).
In addition to difficulties in analysis and time consuming laboratory procedures, it remains desirable for all DNA typing systems to have a higher discriminatory power. Several applications exist in which even the most discriminating tests need improvement in order to remove the considerable remaining doubt resulting from such analyses. Table 6 below lists characteristics of currently available forensic testing systems (Weedn, (1996)) and compares them with the method of the invention.
Applications
As described above, an important application of DNA typing tests is to determine whether a DNA sample (e.g. from a crime scene) originated from an individual suspected of leaving said DNA sample.
There are several applications for DNA typing which require a particularly powerful genotyping system. In a first application, a high powered typing system is advantageous when for example a suspect is identified by searching a DNA profile database such as that maintained by the U.S. Federal Bureau of Investigation. Since databases may contain large numbers of data entries that are expected to increase consistently, currently used forensic systems can be expected to identify several matching DNA profiles due to their relative lack of power. While database searches generally reinforce the evidence by excluding other possible suspects, low powered typing systems resulting in the identification of several individuals may often tend to diminish the overall case against a defendant.
In another application, a target population is systematically tested to identify an individual having the same DNA profile as that of a DNA sample. In such a situation, a defendant is chosen at random based on DNA profile from a large population of innocent individuals. Since the population tested can often be large enough that at least one positive match is identified, and it is usually not possible to exhaustively test a population, the usefulness of the evidence will depend on the level of significance of the forensic test. In order to render such an application useful as a sole or primary source of evidence, DNA typing systems of extremely high discriminatory potential are required.
In yet another application, it is desirable to be able to discriminate between related individuals. Because related individuals will be expected to share a large portion of alleles at polymorphic sites, a very high powered DNA typing assay would be required to discriminate between them. This can have important effects if a sample is found to match the defendant""s DNA profile and no evidence that the perpetrator is a relative can be found.
Accordingly, there is a need in this art for a rapid, simple, inexpensive and accurate technique having a very high resolution value to determine relationships between individuals and differences in degree of relationships. Also, there is a need in the art for a very accurate genetic relationship test procedure which uses very small amounts of an original DNA sample, yet produces very accurate results.
The present invention thus involves methods for the identification of individuals comprising determining the identity of the nucleotides at set of genetic markers in a biological sample, wherein said set of genetic markers comprises at least one DME-related biallelic marker. The present invention provides an extensive set of biallelic markers allowing a higher discriminatory potential than the genetic markers used in current forensic typing systems. Also, biallelic markers can be genotyped in individuals with much higher efficiency and accuracy than the genetic markers used in current forensic typing systems. In preferred embodiments, the invention comprises determining the identity of a nucleotide at a DME-related biallelic marker by single nucleotide primer extension, which does not require electrophoresis as in techniques described above and results in lower rate of experimental error. As shown in Table 6, above, in comparison with PCR based VNTR based methods which allow discriminatory potential of thousands to millions, and RFLP based methods which allow discriminatory potential of merely millions to billions under optimal assumptions, the biallelic marker based method of the present invention provides a radical increase in discriminatory potential.
Any suitable set of genetic markers and biallelic markers of the invention may be used, and may be selected according to the discriminatory power desired. Biallelic markers, sets of biallelic markers, probes, primers, and methods for determining the identity of said biallelic markers are further described herein.
Discriminatory Potential of Biallelic Marker Typing
Calculating Discriminatory Potential
The discriminatory potential of the forensic test can be determined in terms of the profile frequency, also referred to as the random match probability, by applying the product rule. The product rule involves multiplying the allelic frequencies of all the individual alleles tested, and multiplying by an additional factor of 2 for each heterozygous locus.
In one example discussed below, the discriminatory potential of biallelic marker typing can be considered in the context of forensic science. In order to determine the discriminatory potential with respect to the numbers of biallelic markers to be used in a genetic typing system, the formulas and calculations below assume that (1) the population under study is sufficiently large (so that we can assume no consanguinity); (2) all markers chosen are not correlated, so that the product rule (Lander and Budlowle (1992)) can be applied; and (3) the ceiling rule can be applied or that the allelic frequencies of markers in the population under study are known with sufficient accuracy.
As noted in Weir, B. S., Genetic data Analysis II: Methods for Discrete population genetic Data, Sinauer Assoc., Inc., Sunderland, Mass., USA, 1996, the example assumes a crime has been committed and a sample of DNA from the perpetrator (P) is available for analysis. The genotype of this DNA sample can be determined for several genetic markers, and the profile A of the perpetrator can thereby be determined.
In this example, one suspect (S) is available for typing. The same set of genetic markers, such as the biallelic markers of the invention, are typed and the same profile A is obtained for (S) and (P). Two hypotheses are thus presented as follows:
(1) either S is P (event C)
(2) either S is not P (event C).
The ratio L of both probabilities can then be calculated using the following equation:   L  =            pr      ⁡              (                              S            =            A                    ,                      P            =                          A              /              C                                      )                    pr      ⁡              (                              S            =            A                    ,                      P            =                          A              /                              C                _                                                    )            
L can then further be calculated by the following equation:                     L        =                  1                      pr            ⁡                          (                                                P                  =                                                            A                      /                      S                                        =                    A                                                  ,                                  C                  _                                            )                                                                    (          1          )                ⁢                  xe2x80x83                ⁢        Equation        ⁢                  xe2x80x83                ⁢        1            
These probabilities as well as L can be calculated in several settings, notably for different kinship coefficients between P and S for a genetic marker (see Weir, (1996)).
Assuming that all genetic markers chosen are independent of each other, the global ratio L for a set of genetic markers will be the product over each genetic marker of all L.
It is further possible to estimate the mean number of biallelic markers or VNTRs required to have a ratio L equal to 108 or 106 by calculating the expectancy of the random variable L using the following equation:             E      ⁡              (        L        )              =                  ∏                  i          =          1                N            ⁢              xe2x80x83            ⁢                        E          ⁡                      (                          L              i                        )                          ⁢                  xe2x80x83                ⁢        where        ⁢                  xe2x80x83                ⁢        N        ⁢                  xe2x80x83                ⁢        is        ⁢                  xe2x80x83                ⁢        the        ⁢                  xe2x80x83                ⁢        number        ⁢                  xe2x80x83                ⁢        of        ⁢                  xe2x80x83                ⁢        loci                                E        ⁡                  (                      L            i                    )                    =                        ∑                      j            =            1                                G            i                          ⁢                  xe2x80x83                ⁢                              pr            ⁡                          (                                                P                  =                                                                                    A                        ij                                            /                      S                                        =                                          A                      ij                                                                      ,                                  C                  _                                            )                                ·                      L            ij                                ,  
where Aij is the genotype at the ith marker, Lij the ratio associated with such genotype, Gi being the number of genotypes at locus i. From equation 1, it can easily be derived that the expectancy of Li is Gi, the number of possible genotypes of this marker.
The general expectancy for a set of genetic markers can then be expressed by the following equation:                               E          ⁡                      (            L            )                          =                              ∏                          i              =              1                        N                    ⁢                      xe2x80x83                    ⁢                      G            i                                                        (          2          )                ⁢                  xe2x80x83                ⁢        Equation        ⁢                  xe2x80x83                ⁢        2            
Biallelic Marker-based DNA Typing Systems
Using the equations described above, it is possible to select biallelic marker-based DNA typing systems having a desired discriminatory potential.
Using biallelic markers, E(L) can thus be expressed as 3N. When using VNTR-based DNA typing systems, assuming the VNTRs have 10 alleles, E(L) can be expressed as 55N, Based on these results, the number of biallelic markers or VNTRs needed to obtain, in mean, a ratio of at least 106 or 108 can calculated, and are set forth below in Table 7.
Thus, in a first embodiment, DNA typing systems and methods of the invention may comprise genotyping a set of at least 13 or at least 17 biallelic markers to obtain a ratio of at least 106 or 108, assuming a flat distribution of L across the biallelic markers. In preferred embodiments, a greater number of biallelic markers is genotyped to obtain a higher L value. Preferably at least 1, 2, 3, 4, 5, 10, 13, 15, 17, 20, 25, 30, 40, 50, 70, 85, 100, 150, or all of the DME-related biallelic markers are genotyped. Said DNA typing systems of the invention would result in L values as listed in Table 8 below as an indication of the discriminate potential of the systems of the invention.
In situations where the distribution of L is not flat, such as in the worst case when the perpetrator is homozygous for the major allele at each genetic locus and L thus takes the lowest value, a larger number of biallelic markers is required for the same discriminatory potential. Therefore, in preferred embodiments, DNA typing systems and methods of the invention using a larger number of biallelic markers allow for uneven distributions of L across the biallelic markers. For example, assuming unrelated individuals, a set of independent markers having an allelic frequency of 0.1/0.9, and the genetic profile of a homozygote at each genetic loci for the major allele, 66 biallelic markers are required to obtain a ratio of 106, and 88 biallelic markers are required to obtain a ratio of 108. Thus, in preferred embodiments based on the use of markers having a major allele of sufficiently high frequency, this is a first estimation of the upper bound of markers required in a DNA typing system.
In further embodiments, it is also desirable to have the ability to discriminate between relatives. Although unrelated individuals have a low probability of sharing genetic profiles, the probability is greatly increased for relatives. For example, the DNA profile of a suspect matches the DNA profile of a sample at a crime scene, and the probability of obtaining the same DNA profile if left by an untyped relative is required. Table 9 below (Weir (1996)) lists probabilities for several different types of relationships, assuming alleles Ai and Aj, and population frequencies pi and pj, and lists likelihood ratios assuming genetic loci having allele frequencies of 0.1.
In one example, where the suspect is the full brother of the perpetrator, the number of required biallelic markers will be 187 assuming the profile is that of a homozygote for the major allele at each biallelic marker.
In yet further embodiments, the DNA typing systems and methods of the present invention may further take into account effects of subpopulations on the discriminatory potential. In embodiments described above for example, DNA typing systems consider close familial relationships, but do not take into account membership in the same population. While population membership is expected to have little effect, the invention may further comprise genotyping a larger set of biallelic markers to achieve higher discriminatory potential. Alternatively, a larger set of biallelic markers may be optimized for typing selected populations; alternatively, the ceiling principle may be used to study allele frequencies from individuals in various populations of interest, taking for any particular genotype the maximum allele frequency found among the populations.
The invention thus encompasses methods for genotyping comprising determining the identity of a nucleotide at least 13, 15, 17, 20, 25, 30, 40, 50, 66, 70, 85, 88, 100, 187, 200, 300, 500, 700, 1000 or 2000 biallelic markers in a biological sample, wherein at least 1, 2, 3, 4, 5, 10, 13, 17, 20, 25, 30, 40, 50, 70, 85, 100, 150, 200, 300, 400 or all of said biallelic markers are DME-related biallelic markers selected from the group consisting of SEQ ID Nos. 485-487, 494-531, 533-547, 549-846, 848-956, 958-977.
Any markers known in the art may be used with the DME-related biallelic markers of the present invention in the DNA typing methods and systems described herein, for example in anyone of the following web sites offering collections of SNPs and information about those SNPs:
The Genetic Annotation Initiative (web site: cgap.nci.nih.gov/GAI/). An NIH run site which contains information on candidate SNPs thought to be related to cancer and tumorigenesis generally.
dbSNP Polymorphism Repository (world wide web site: ncbi.nlm.nih.gov/SNP/). A more comprehensive NIH-run database containing information on SNPs with broad applicability in biomedical research.
HUGO Mutation Database Initiative (web site: ariel.ucs.unimelb.edu.au:80/xcx9ccotton/mdi.htm). A database meant to provide systematic access to information about human mutations including SNPs. This site is maintained by the Human Genome Organisation (HUGO).
Human SNP Database (world wide web site: -genome.wi.mit.edu/SNP/human/index.html). Managed by the Whitehead Institute for Biomedical Research Genome Institute, this site contains information about SNPs resulting from the many Whitehead research projects on mapping and sequencing.
SNPs in the Human-Genome SNP database (world wide web site: ibc.wustl.edu/SNP). This website provides access to SNPs that have been organized by chromosomes and cytogenetic location. The site is run by Washington University.
HGBase (web site: hgbase.cgr.ke.se/). HGBASE is an attempt to summarize all known sequence variations in the human genome, to facilitate research into how genotypes affect common diseases, drug responses, and other complex phenotypes, and is run by the Karolinska Institute of Sweden.
The SNP Consortium Database (web site: snp.cshl.org/db/snp/map). A collection of SNPs and related information resulting from the collaborative effort of a number of large pharmaceutical and information processing companies.
GeneSNPs (world wide web site: genome.utah.edu/genesnps/). Run by the University of Utah, this site contains information about SNPs resulting from the U.S. National Institute of Environmental Health""s initiative to understand the relationship between genetic variation and response to environmental stimuli and xenobiotics.
In addition, biallelic markers provided in the following patents and patent applications may also be used with the map-realted biallelic markers of the invention in the DNA typing methods and systems described above: U.S. Serial No. 60/206,615, filed Mar. 24, 2000; U.S. Serial No. 60/216,745, filed Jun. 30, 2000; WIPO Serial No. PCT/IB00/00184, filed Feb. 11, 2000; WIPO Serial No. PCT/IB98/01193, filed Jul. 17, 1998; PCT Publication No. WO 99/54500, filed Apr. 21, 1999; and WIPO Serial No. PCT/IB00/00403, filed Mar. 24, 2000.
Biallelic markers, sets of biallelic markers, probes, primers, and methods for determining the identity of a nucleotide at said biallelic markers are also encompassed and are further described herein, and may encompass any further limitation described in this disclosure, alone or in any combination.
Forensic matching by microsequencing is further described in Example 7 below.